Anti-c-met/anti-ang2 bispecific antibody

ABSTRACT

An anti-c-Met/anti-Ang2 bispecific antibody, a composition including the anti-c-Met/anti-Ang2 bispecific antibody, and a method of preventing and/or treating a cancer in a subject including administering the anti-c-Met/anti-Ang2 bispecific antibody to the subject.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of Korean Patent Application No.10-2013-0147624 filed on Nov. 29, 2013 in the Korean IntellectualProperty Office, the entire disclosure of which is hereby incorporatedby reference.

INCORPORATION-BY-REFERENCE OF MATERIAL ELECTRONICALLY SUBMITTED

Incorporated by reference in its entirety herein is a computer-readablenucleotide/amino acid sequence listing submitted herewith and identifiedas follows: One 186,084 bytes ASCII (Text) file named “718895_ST25.TXT”created Dec. 1, 2014.

BACKGROUND OF THE INVENTION

1. Field

Provided is an anti-c-Met/anti-Ang2 bispecific antibody, a compositionincluding the anti-c-Met/anti-Ang2 bispecific antibody, and a method ofpreventing and/or treating a cancer in a subject including administeringthe anti-c-Met/anti-Ang2 bispecific antibody to the subject.

2. Description of the Related Art

Angiopoietin2 (Ang2) is an antagonistic ligand of receptor Tie2 presentin vascular endothelial cell (Nat Rev Mol Cell Biol. 2009 March;10(3):165-77), and competes with Angiopoietin1 (Ang1) which is anagonist of Tie2 for binding to Tie2, thereby inhibiting the signaltransduction by Tie2 (Science. 1997 Jul. 4; 277(5322):55-60). Therefore,Ang2 inhibits Ang1-Tie2 signal transduction for maintaining stability ofvascular endothelial cells, leading to stimulation of angiogenesis bydynamic rearrangement of blood vessels (Science. 1997 Jul. 4;277(5322):48-50). It is known from many preclinical studies that sincethe angiogenesis process is essential to cancer growth, the inhibitionof the Tie2-dependent Ang2 functions can lead to the inhibition ofangiogenesis, thereby preventing additional growth of cancer (J Natl.Cancer Inst. 2012 Mar. 21; 104(6):461-75). There have been many attemptsto prevent cancer using an Ang2 specific antibody by many globalpharmaceutical companies, such as Regeneron, Astrazeneca, Amgen, and thelike. However, the recent studies have suggested that when a cancer cellis treated with an angiogenesis inhibitor, a mechanism for avoiding thesudden oxygen deficiency condition is activated in the cancer cell,which can stimulate cancer metastasis (Nat. Rev. Clin. Oncol. 2011 Mar.1; 8(4):210-21) even in the absence of angiogenesis. Therefore, to avoidsuch serious side effects of angiogenesis inhibitors, it is necessary toinhibit functions of cancer metastasis-related proteins as well.

c-Met is a representative receptor tyrosine kinase (RTK) present oncertain cell surfaces. c-Met binds to its ligand, Hepatocyte GrowthFactor/Scattering Factor (HGF/SF), to promote intracellular signaltransduction, thereby stimulating cell growth, and it is overexpressedin many cancer cells, thereby widely relating to cancer occurrence,cancer metastasis, cancer cell migration, cancer cell invasion, andangiogenesis. In addition, c-Met is a representative early protein ofcancer metastasis, because c-Met signaling through HGF/SF weakenscell-cell contact in almost all types of epithelial tumors, leading toscattering. (Nat Rev Cancer. 2012 Jan. 24; 12(2):89-103). In particular,hypoxia-response elements are present at the upstream of c-Met gene, andthe expression of the gene is increased under oxygen deficientconditions (Oral Oncol. 2006 July; 42(6):593-8).

Therefore, simultaneous inhibition of Ang2 and c-Met may lead to moreeffective inhibition of cancer cell growth and metastasis, and thus, itis needed to develop a drug that simultaneously targets both Ang2 andc-Met.

BRIEF SUMMARY OF THE INVENTION

One embodiment provides an anti-c-Met/anti-Ang2 bispecific antibodyincluding (a) an anti-c-Met antibody or an antigen-binding fragmentthereof and (b) an anti-Ang2 antibody or an antigen-binding fragmentthereof.

The anti-c-Met antibody or an antigen-binding fragment thereof may be anantibody or an antigen-binding fragment thereof which specifically bindsto an epitope including or consisting essentially of 5 or moreconsecutive amino acids in SEMA domain of c-Met protein; and theanti-Ang2 antibody or an antigen-binding fragment thereof may be anantibody or an antigen-binding fragment thereof which specifically bindsto (1) at least one amino acid residue selected from the groupconsisting of amino acid residues exposed to the outside of loop 1, loop2, and loop 3 of human Ang2 on its tertiary structure or (2) an aminoacid sequence region including about 2 to about 20 consecutive aminoacids on the tertiary structure of human Ang2 including the at the leastone amino acid residue.

Another embodiment provides a pharmaceutical composition including theanti-c-Met/anti-Ang2 bispecific antibody.

Another embodiment provides a method of preventing and/or treating acancer in a subject including administering the anti-c-Met/anti-Ang2bispecific antibody to the subject.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates is a schematic showing a structure of ananti-c-Met/anti-Ang2 bispecific antibody according to an embodiment.

FIG. 2 is a graph showing the inhibition of binding between Ang2 andintegrin by treatment of an anti-Ang2 antibody according to anembodiment (control antibody 1: Regeneron Ang2 antibody; controlantibody 2: Astra Zeneca Ang2 antibody).

FIG. 3 is a graph showing the binding affinity of ananti-c-Met/anti-Ang2 bispecific antibody according to an embodiment toAng2.

FIG. 4 is a graph showing the inhibition of cancer cell (HUVEC) growthby treatment of an anti-c-Met/anti-Ang2 bispecific antibody according toan embodiment.

FIG. 5 is a graph showing the inhibition of cancer cell (LEC) growth bytreatment of an anti-c-Met/anti-Ang2 bispecific antibody according to anembodiment.

FIG. 6 is a graph showing the inhibition of cancer cell metastasis bytreatment of an anti-c-Met/anti-Ang2 bispecific antibody according to anembodiment.

DETAILED DESCRIPTION OF THE INVENTION

The pre-existing targeting drugs recognizing only Ang2 in cancer cellsoften induces over-expression and mutation of c-Met in a cancer cellthereby making the cancer cell to acquire a resistance thereto, leadingto decreasing the therapeutic effect thereof. A bispecific antibodysimultaneously recognizing c-Met and Ang2 can block a signaltransduction by c-Met in a cancer cell, thereby preventing thegeneration of a drug resistance in a subject, and thus, it can exhibitan excellent cancer cell inhibitory effect even in a cancer cell havinga drug resistance.

An embodiment provides an anti-c-Met/anti-Ang2 bispecific antibodyincluding (a) an anti-c-Met antibody or an antigen-binding fragmentthereof and (b) an anti-Ang2 antibody or an antigen-binding fragmentthereof. The bispecific antibody can simultaneously recognize and bindto c-Met and Ang2, and inhibit the functions thereof, thereby exhibitingsynergistic anti-cancer effects.

As used herein, the term “antibody” refers to all substance generated byan antigen stimulation in immune system, and has no specific limitationin its type. The antibody may include animal antibodies, chimericantibodies, humanized antibodies, or human antibodies. In addition, theantibody may cover any antigen-binding fragment possessing antigenbinding capacity. The term “complementarity-determining regions (CDR)”may refer to a part of a variable region of an antibody, which allows abinding specificity to a specific antigen. The term “antigen-bindingfragment” may refer to any antibody fragment including at least one thecomplementarity-determining region, and for example, it may be selectedfrom the group consisting of scFv, (scFv)₂, scFv-Fc, Fab, Fab′, andF(ab′)₂.

In the following description for the anti-c-Met antibody or anantigen-binding fragment thereof and the anti-Ang2 antibody or anantigen-binding fragment thereof, the regions except for heavy chainCDRs and light chain CDRs, or a heavy chain variable region and a lightchain variable region may be originated (obtained) from any subtype ofimmunoglobulin (e.g., IgA, IgD, IgE, IgG (IgG1, IgG2, IgG3, IgG4), IgM,etc.), and for example, may be originated (obtained) from a light chainconstant region and/or a heavy chain constant region of any subtype ofimmunoglobulin.

“c-Met” or “c-Met protein”, a target of a bispecific antibody to beprovided in one embodiment, refers to a receptor tyrosine kinase (RTK)which binds hepatocyte growth factor (HGF). c-Met may be derived(obtained) from any species, particularly a mammal, for instance,primates such as human c-Met (e.g., NP_000236), monkey c-Met (e.g.,Macaca mulatta, NP_001162100), or rodents such as mouse c-Met (e.g.,NP_032617.2), rat c-Met (e.g., NP_113705.1), or the like. The c-Metprotein may include a polypeptide encoded by the nucleotide sequenceidentified as GenBank Accession Number NM_000245, a polypeptide havingthe amino acid sequence identified as GenBank Accession Number NP_000236or extracellular domains thereof. The receptor tyrosine kinase c-Metparticipates in various biological processes, such as cancer incidence,metastasis, migration of cancer cell, invasion of cancer cell,angiogenesis, and the like.

Ang2, another target of a bispecific antibody to be provided in oneembodiment, is closely related to angiogenesis. It is a soluble ligandpresent in blood, and it is widely involved in angiogenesis, metastasis,and cancer cell invasion. The Ang2 may be derived (obtained) frommammals including primates such as humans and monkeys and rodents suchas rats and mice and for example, it may be selected from the groupconsisting of a human Ang2 (e.g., NCBI Accession #O15123), a monkey Ang2(e.g., NCBI Accession No. Q8MIK6 etc.), a mouse Ang2 (NCBI Accession#NP_031452, Accession #O35608, etc.), a rat Ang2 (e.g., NCBI AccessionNo. O35462, etc.), and any combination thereof.

The anti-c-Met/anti-Ang2 bispecific antibody can inhibit the bindingbetween Ang2 and Tie-2 receptor by competing with Tie2 in binding toAng2, and for example, it may inhibit binding between Ang2 and Tie2receptor by recognizing and/or binding to a binding site of Ang2 forbinding to Tie2 receptor. The anti-Ang2 antibody or an antigen-bindingfragment thereof may recognize human Ang2 which widely relates toangiogenesis, cancer metastasis, and cancer cell invasion, and inhibitsbinding of Ang2 to its receptor, Tie-2 receptor, thereby inhibitingangiogenesis, cancer generation and cancer metastasis, and exhibitingtreating and/or preventing effects on a disease associated withover-activation and/or over-expression of Ang2.

Furthermore, the anti-Ang2 antibody or an antigen-binding fragmentthereof may further inhibit binding between Ang2 and integrin. Integrinis a typical protein which mediates cell adhesion and has a heterodimerstructure including an alpha (α) subunit and a beta (β) subunit. Inmammals, 18 types of alpha subunits and 8 types of beta subunits havebeen identified. The integrin may be derived from mammals includingprimates such as humans and monkeys and rodents such as mice and ratsand for example, it may be a human integrin, a monkey integrin, a mouseintegrin and a rat integrin, but is not limited thereto. In each andevery species, 24 types of integrins are known and amino acid sequencesthereof have been well identified such that they are clearly known thosewho have ordinary knowledge in the art For example, the integrin may bea human integrin and typical human integrin types may include, but notlimited to, alpha5beta1 (α5β1) (α5: NCBI Accession No. P08648, (β1: NCBIAccession No. P05556), alphaVbeta1 (αVβ1) (αV: NCBI Accession No.P06756, β1: NCBI Accession No. P05556), and alphaVbeta3 (αVβ3) (αV: NCBIAccession No. P06756, β3: NCBI Accession No. P05106).

As described above, in addition to the inhibitory effect on the bindingof Ang2 and Tie2 receptor, the anti-Ang2 antibody or an antigen-bindingfragment thereof further inhibits the binding between Ang2 and integrinthat is another protein involved in cancer cell growth and/ormetastasis, thereby having more increased inhibition effects on thecancer cell growth and/or metastasis, and being capable of exhibitingsuch inhibition effects even in a cell where Tie2 is nor expressed.

The anti-Ang2 antibody or an antigen-binding fragment thereof used in anembodiment may be described as follows. The anti-Ang2 antibody or anantigen-binding fragment thereof may recognize all or part (for example,at least one amino acid selected from the group consisting of the aminoacid residue regions exposed to the outside of each loop) of the regionsconsisting of loop 1 (a region covering from 417^(th) to 434^(th) aminoacids of SEQ ID NO: 172), loop 2 (a region covering from 447^(th) to454^(th) amino acids of SEQ ID NO: 172), and loop 3 (a region coveringfrom 460^(th) to 468^(th) amino acids of SEQ ID NO: 172) of human Ang2(hAng2; SEQ ID NO: 172; Accession #O15123), or an amino acid sequenceregion including about 2 to about 20, about 2 to about 15, about 2 toabout 10, or about 2 to about 5 consecutive amino acids within SEQ IDNO: 172 including at least one exposed amino acid residue of loop 1,loop 2, or loop 3 of SEQ ID NO: 172, as an epitope, or specifically bindthereto. An “exposed” amino acid is an amino acid that is exposed tosolution and available for binding when a protein (e.g., Ang2) is in itsnative conformation in a biological medium or other solution underphysiological conditions (e.g., physiological pH, isotonicity,temperature, etc.)

For example, the anti-Ang2 antibody or an antigen-binding fragmentthereof may recognize at least one amino acid residue selected from thegroup consisting of I434 positioned at loop 1, A449 and P452 positionedat loop 2, N467 positioned at loop 3 of human Ang2, and any combinationthereof, or an amino acid sequence region including about 2 to about 20,about 2 to about 15, about 2 to about 10, or about 2 to about 5consecutive amino acids including at least one of the above-describedamino acid residues from loop 1, 2, or 3 as an epitope and/orspecifically bind thereto. In one embodiment, the anti-Ang2 antibody oran antigen-binding fragment thereof may recognize an epitope comprisingat least one amino acid residue selected from the group consisting ofI434 positioned at loop 1, A449 and P452 positioned at loop 2, and N467positioned at loop 3 of human Ang2, or specifically bind to thisportion.

[Ang2 (SEQ ID NO: 172; loop 1, loop 2 and loop 3 are underlinedrespectively in order and each epitope is marked in bold and italicletters)]

MWQIVFFTLS CDLVLAAAYN NFRKSMDSIG KKQYQVQHGSCSYTFLLPEM DNCRSSSSPY VSNAVQRDAP LEYDDSVQRLQVLENIMENN TQWLMKLENY IQDNMKKEMV EIQQNAVQNQTAVMIEIGTN LLNQTAEQTR KLTDVEAQVL NQTTRLELQLLEHSLSTNKL EKQILDQTSE INKLQDKNSF LEKKVLAMEDKHIIQLQSIK EEKDQLQVLV SKQNSIIEEL EKKIVTATVNNSVLQKQQHD LMETVNNLLT MMSTSNSAKD PTVAKEEQISFRDCAEVFKS GHTTNGIYTL TFPNSTEEIK AYCDMEAGGGGWTIIQRRED GSVDFQRTWK EYKVGFGNPS GEYWLGNEFVSQLTNQQRYV LKIHLKDWEG NEAYSLYEHF YLSSEELNYRIHLKGLTGTA GKISSISQPG NDFSTKDGDN DKCI CKCSQMLTGGWWFDAC GPSNLNGMYY PQRQNTNKFN GIKWYYWKGS GYSLKATTMM IRPADF

The above epitope sites are exposed amino acid residues positioned atloop 1, loop 2, or loop 3 of the three dimensional structure of Ang2,and they directly participate in binding with a Tie2 receptor or theyare positioned by being included in the binding site with the Tie2receptor or neighboring thereupon (see FIG. 1). Accordingly, theanti-Ang2 antibody or an antigen-binding fragment thereof recognizingand binding to the above at least one epitope competes with the Tie2receptor in binding to Ang2 and thus, inhibits binding between Ang2 andthe Tie2 receptor.

The term “consecutive amino acid” may include amino acids which areadjacent to one another on the secondary or tertiary structure of aprotein as well as amino acids which are continuous on their primaryamino acid sequences. Accordingly, the “consecutive amino acid residues”as used herein may refer to contiguous amino acid residues on theprimary, secondary, or tertiary structure of a protein.

Embodiments of the invention also include an antibody or anantigen-binding fragment thereof that competes with the anti-Ang2antibody for binding to Ang2, or that competes with the Tie2 receptor inbinding to Ang2, and can inhibit binding between Ang2 and the Tie2receptor. This competitive-binding antibody may be an antibodyrecognizing a site adjacent to the aforementioned epitopes on its threedimensional structure of Ang2 as an epitope. The competitive-bindingantibody may have a binding affinity (Kd) of about 0.001 to about 10 nM,about 0.01 to about 1 nM, or about 0.1 to about 0.8 nM.

Therefore, the anti-Ang2 antibody or an antigen-binding fragment thereofmay be at least one selected from the group consisting of an antibody oran antigen-binding fragment thereof recognizing and/or specificallybinding to the aforementioned epitope, and an antibody competingtherewith for binding to Ang2 or an antigen-binding fragment thereof.

In a particular embodiment, the anti-Ang2 antibody or an antigen-bindingfragment thereof may include or consist essentially of, as a heavy chaincomplementarity determining region (CDR), at least one selected from thegroup consisting of a polypeptide (CDR-H1) including the amino acidsequence of SEQ ID NO: 151, a polypeptide (CDR-H2) including the aminoacid sequence of SEQ ID NO: 152 and a polypeptide (CDR-H3) including anamino acid sequence selected from the group consisting of SEQ ID NOs:122 to 129, for example, an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 122 to 127:

(SEQ ID NO: 151) X₁-Y-X₂-M-S

wherein,

X₁ is aspartic acid (D), serine (S), or asparagine (N), for example,aspartic acid (D) or asparagine (N), and

X₂ is alanine (A), aspartic acid (D), or tyrosine (Y); and

(SEQ ID NO: 152) X₃-I-X₄-X₅-X₆-X₇-X₈-X₉-X₁₀-Y-Y-A-D-S-V-K-G

wherein,

X₃ is alanine (A), glycine (G), leucine (L), or serine (S), for example,alanine (A), glycine (G), or serine (S),

X₄ is tyrosine (Y) or serine (S),

X₅ is proline (P), histidine (H), or serine (S),

X₆ is aspartic acid (D), glycine (G), or serine (S),

X₇ is serine (S), glycine (G), or aspartic acid (D),

X₈ is glycine (G) or serine (S),

X₉ is asparagine (N) or serine (S), and

X₁₀ is lysine (K), isoleucine (I), or threonine (T).

The anti-Ang2 antibody or an antigen-binding fragment thereof mayinclude or consist essentially of, as a light chain complementaritydetermining region (CDR), at least one selected from the groupconsisting of a polypeptide (CDR-L1) including the amino acid sequenceof SEQ ID NO: 153, a polypeptide (CDR-L2) including the amino acidsequence of SEQ ID NO: 154, and a polypeptide (CDR-L3) including theamino acid sequence of SEQ ID NO: 155:

(SEQ ID NO: 153) X₁₁-G-S-S-S-N-I-G-X₁₂-N-X₁₃-V-X₁₄

wherein,

X₁₁ is serine (S) or threonine (T),

X₁₂ is asparagine (N) or serine (S),

X₁₃ is alanine (A), tyrosine (Y), or aspartic acid (D), and

X₁₄ is asparagine (N), serine (S), threonine (T), or tyrosine (Y);

(SEQ ID NO: 154) X₁₅-X₁₆-X₁₇-X₁₈-R-P-S

wherein,

X₁₅ is alanine (A) or serine (S),

X₁₆ is aspartic acid (D) or asparagine (N),

X₁₇ is serine (S) or asparagine (N), for example serine (S), and

X₁₈ is asparagine (N), Lysine (K), histidine (H), or glutamine (Q); and

(SEQ ID NO: 155) X₁₉-X₂₀-W-D-X₂₁-S-L-X₂₂-X₂₃

wherein,

X₁₉ is glycine (G) or alanine (A),

X₂₀ is serine (S), alanine (A), or threonine (T), for example serine (S)or threonine (T),

X₂₁ is tyrosine (Y) or aspartic acid (D), for example, tyrosine (Y),

X₂₂ is serine (S) or asparagine (N), for example, serine (S), and

X₂₃ is glycine (G) or alanine (A).

In an embodiment, the anti-Ang2 antibody or an antigen-binding fragmentthereof may include or consist essentially of the above heavy chaincomplementarity determining regions, light chain complementaritydetermining regions, or any combination thereof.

More particularly, the anti-Ang2 antibody or an antigen-binding fragmentthereof may include or consist essentially of:

at least one heavy chain complementarity determining region selectedfrom the group consisting of a polypeptide (CDR-H1) including the aminoacid sequence of SEQ ID NO: 151, a polypeptide (CDR-H2) including theamino acid sequence of SEQ ID NO: 152, and a polypeptide (CDR-H3)including an amino acid sequence selected from the group consisting ofSEQ ID NOs: 122 to 129, for example, an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 122 to 127, or a heavy chainvariable region including the at least one heavy chain complementaritydetermining region;

at least one light chain complementarity determining region selectedfrom the group consisting of a polypeptide (CDR-L1) including the aminoacid sequence of SEQ ID NO: 153, a polypeptide (CDR-L2) including theamino acid sequence of SEQ ID NO: 154, and a polypeptide (CDR-L3)including the amino acid sequence of SEQ ID NO: 155, or a light chainvariable region including the at least one light chain complementaritydetermining region;

a combination of the at least one heavy chain complementaritydetermining region and the at least one light chain complementaritydetermining region; or

a combination of the heavy chain variable region and the light chainvariable region.

For example, the heavy chain CDR of the anti-Ang2 antibody may includeor consist essentially of the amino acid sequence as set forth infollowing Table 1.

TABLE 1 Amino acid sequence of heavy chain CDR CDRH1-KABAT CDRH2-KABATCDRH3-KABAT DYAMS AIYPDSGNKYYADSVK ARHSSDPKVKSGYYD (SEQ ID NO: G DGMDV109) (SEQ ID NO: 115) (SEQ ID NO: 122) DYYMS GIYPSGGSTYYADSVKARDPSTLTYAGFDY (SEQ ID NO: G (SEQ ID NO: 123) 110) (SEQ ID NO: 116)NYAMS AISSGGGNIYYADSVK AKSGIQPSPPSMSSA (SEQ ID NO: G YAMDV 111)(SEQ ID NO: 117) (SEQ ID NO: 124) DYAMS SIYPDDGNTYYADSVK ARHTSHHTSIDGYYY(SEQ ID NO: G YGMDG 109) (SEQ ID NO: 118) (SEQ ID NO: 125) DYDMSSISHGDSNKYYADSVK AKSSGIQESPPTYYY (SEQ ID NO: G YGMDV 112)(SEQ ID NO: 119) (SEQ ID NO: 126) DYAMS SIYPDDGNTYYADSVK AKHPVRLNLHPMYYY(SEQ ID NO: G YGMDV 109) (SEQ ID NO: 118) (SEQ ID NO: 127) SYDMSLISPDSSSIYYADSVK AKDLISFWRGGFDY (SEQ ID NO: G (SEQ ID NO: 128) 113)(SEQ ID NO: 120) DYDMS GISSDDGNTYYADSVK ARPTIDKYTLRGYYS (SEQ ID NO: GYGMDV 114) (SEQ ID NO: 121) (SEQ ID NO: 129)

In addition, the light chain CDR of the anti-Ang2 antibody may includeor consist essentially of the amino acid sequence as set forth infollowing Table 2.

TABLE 2 Amino acid sequence of light chain CDR CDRL1-KABAT CDRL2-KABATCDRL3-KABAT SGSSSNIGNNAVN ADSNRPS GSWDYSLSG (SEQ ID NO: (SEQ ID NO:(SEQ ID NO: 130) 138) 145) SGSSSNIGNNYVT ADSHRPS ATWDYSLSG (SEQ ID NO:(SEQ ID NO: (SEQ ID NO: 131) 139) 146)  SGSSSNIGNNDVY ANSHRPS GTWDYSLSG(SEQ ID NO: (SEQ ID NO: (SEQ ID NO: 132) 140) 147) TGSSSNIGNNDVS SDSKRPSGSWDYSLSG (SEQ ID NO: (SEQ ID NO: (SEQ ID NO: 133) 141) 145)SGSSSNIGSNAVN ADSNRPS GSWDYSLSG (SEQ ID NO: (SEQ ID NO: (SEQ ID NO: 134)138) 145) TGSSSNIGNNAVS SDSQRPS ATWDYSLSA (SEQ ID NO: (SEQ ID NO:(SEQ ID NO: 135) 142) 148) SGSSSNIGSNYVN SDSHRPS GAWDDSLSG (SEQ ID NO:(SEQ ID NO: (SEQ ID NO: 136) 143) 149) TGSSSNIGSNYVS SDNKRPS GTWDDSLNG(SEQ ID NO: (SEQ ID NO: (SEQ ID NO: 137) 144) 150)

In particular, the anti-Ang2 antibody or an antigen-binding fragmentthereof may include or consist essentially of:

at least one heavy chain complementarity determining region selectedfrom the group consisting of a polypeptide (CDR-H1) including an aminoacid sequence selected from SEQ ID NOs: 109 to 114, for example, anamino acid sequence selected from SEQ ID NOs: 109 to 112, a polypeptide(CDR-H2) including an amino acid sequence selected from SEQ ID NOs: 115to 121, for example, an amino acid sequence selected from SEQ ID NOs:115 to 119, and a polypeptide (CDR-H3) including an amino acid sequenceselected from the group consisting of SEQ ID NOs: 122 to 129, forexample, an amino acid sequence selected from SEQ ID NOs: 122 to 127, ora heavy chain variable region including the at least one heavy chaincomplementarity determining region;

at least one light chain complementarity determining region selectedfrom the group consisting of a polypeptide (CDR-L1) including an aminoacid sequence selected from SEQ ID NOs: 130 to 137, for example, anamino acid sequence selected from SEQ ID NOs: 130 to 135, a polypeptide(CDR-L2) including an amino acid sequence selected from SEQ ID NOs: 138to 144, for example, an amino acid sequence selected from SEQ ID NOs:138 to 142, and a polypeptide (CDR-L3) including an amino acid sequenceselected from SEQ ID NOs: 145 to 150, for example, an amino acidsequence selected from SEQ ID NOs: 145 to 148, or a light chain variableregion including the at least one light chain complementaritydetermining region;

a combination of the at least one heavy chain complementaritydetermining region and the at least one light chain complementaritydetermining region; or

a combination of the heavy chain variable region and the light chainvariable region.

In the anti-Ang2 antibody or an antigen-binding fragment thereof, theheavy chain variable region may include or consist essentially of anamino acid sequence selected from the group consisting of SEQ ID NOs:156 to 163, for example an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 156 to 161, and the light chain variableregion may include or consist essentially of an amino acid sequenceselected from the group consisting of SEQ ID NOs: 164 to 171, forexample an amino acid sequence selected from the group consisting of SEQID NOs: 164 to 169.

Accordingly, the anti-Ang2 antibody or an antigen-binding fragmentthereof may include or consist essentially of a heavy chain variableregion including an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 156 to 163, for example an amino acid sequenceselected from the group consisting of SEQ ID NOs: 156 to 161; a lightchain variable region including an amino acid sequence selected from thegroup consisting of SEQ ID NO: 164 or 171, for example an amino acidsequence selected from the group consisting of SEQ ID NOs: 164 to 169;or a combination of the heavy chain variable region and the light chainvariable region.

The amino acid sequences of the heavy chain variable region and thelight chain variable region of the anti-Ang2 antibody according to anembodiment are summarized in Table 3:

TABLE 3 Amino acid sequence of heavy Amino acid sequence of chainvariable region light chain variable regionEVQLLESGGGLVQTGGSLRLSCAASGFTFSDYA QSVLTQPPSASGTPGQRVTISCSGSSSNIGNNAVMSWVRQAPGKGLEWVSAIYPDSGNKYYADSV NWYQQLPGTAPKLLIYADSNRPSGVPDRFSGSKKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC SGTSASLAISGLRSEDEADYYCGSWDYSLSGYVARHSSDPKVKSGYYDDGMDVWGQGTLVAVS FGGGTKLTVLG (SEQ ID NO: 164) S (SEQ IDNO: 156) EVQLLESGGGLVQPGGSLRLSCAASGFTFSDYYQSVLTQPPSASGTPGQRVTISCSGSSSNIGNNYV MSWVRQAPGKGLEWVSGIYPSGGSTYYADSVTWYQQLPGTAPKLLIYADSHRPSGVPDRFSGSK KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCSGTSASLAISGLRSEDEADYYCATWDYSLSGY ARDPSTLTYAGFDYWGQGTLVTVSS (SEQ IDVFGGGTKLTVLG (SEQ ID NO: 165) NO: 157) EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYAQSVLTQPPSASGTPGQRVTISCSGSSSNIGNNDV MSWVRQAPGKGLEWVSAISSGGGNIYYADSVYWYQQLPGTAPKLLIYANSHRPSGVPDRFSGSK KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCSGTSASLAISGLRSEDEADYYCGTWDYSLSGY AKSGIQPSPPSMSSAYAMDVWGQGTLVTVSSVFGGGTKLTVLG (SEQ ID NO: 166) (SEQ ID NO: 158)EVQLLESGGGLVQPGGSLRLSCAASGFTFSDYA QSVLTQPPSASGTPGQRVTISCTGSSSNIGNNDVMSWVRQAPGKGLEWVSSIYPDDGNTYYADSV SWYQQLPGTAPKLLIYSDSKRPSGVPDRFSGSKKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC SGTSASLAISGLRSEDEADYYCGSWDYSLSGYVARHTSHHTSIDGYYYYGMDGWGQGTLVTVSS FGGGTKLTVLG (SEQ ID NO: 167) (SEQ ID NO:159) EVQLLESGGGLVQPGGSLRLSCAASGFTFSDYDQSVLTQPPSASGTPGQRVTISCSGSSSNIGSNAV MSWVRQAPGKGLEWVSSISHGDSNKYYADSVNWYQQLPGTAPKLLIYADSNRPSGVPDRFSGSK KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCSGTSASLAISGLRSEDEADYYCGSWDYSLSGYV AKSSGIQESPPTYYYYGMDVWGQGTLVTVSSFGGGTKLTVLG (SEQ ID NO: 168) (SEQ ID NO: 160)EVQLLESGGGLVQTGGSLRLSCAASGFTFSDYA QSVLTQPPSASGTPGQRVTISCTGSSSNIGNNAVMSWVRQAPGKGLEWVSSIYPDDGNTYYADSV SWYQQLPGTAPKLLIYSDSQRPSGVPDRFSGSKKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC SGTSASLAISGLRSEDEADYYCATWDYSLSAYVAKHPVRLNLHPMYYYYGMDVWGQGTLVTVS FGGGTKLTVLG (SEQ ID NO: 169) S (SEQ IDNO: 161) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYDQSVLTQPPSASGTPGQRVTISCSGSSSNIGSNYV MSWVRQAPGKGLEWVSLISPDSSSIYYADSVKNWYQQLPGTAPKLLIYADSNRPSGVPDRFSGSK GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASGTSASLAISGLRSEDEADYYCGSWDYSLSGYV KDLISFWRGGFDYWGQGTLVTVSS (SEQ IDFGGGTKLTVLG (SEQ ID NO: 170) NO: 162) EVQLLESGGGLVQPGGSLRLSCAASGFTFSDYDQSVLTQPPSASGTPGQRVTISCTGSSSNIGSNYV MSWVRQAPGKGLEWVSGISSDDGNTYYADSVSWYQQLPGTAPKLLIYSDNKRPSGVPDRFSGSK KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCSGTSASLAISGLRSEDEADYYCGTWDDSLNGY ARPTIDKYTLRGYYSYGMDVWGQGTLVTVSSVFGGGTKLTVLG (SEQ ID NO: 171) (SEQ ID NO: 163)

In another embodiment, the anti-Ang2 antibody or an antigen-bindingfragment thereof may include or consist essentially of:

at least one heavy chain complementarity determining region selectedfrom the group consisting of a polypeptide (CDR-H1) including an aminoacid sequence of SEQ ID NO: 113 or 114; a polypeptide (CDR-H2) includingan amino acid sequence of SEQ ID NO: 120 or 121, and a polypeptide(CDR-H3) including an amino acid sequence of SEQ ID NO: 128 or 129, or aheavy chain variable region including the at least one heavy chaincomplementarity determining region;

at least one light chain complementarity determining region selectedfrom the group consisting of a polypeptide (CDR-L1) including an aminoacid sequence of SEQ ID NO: 136 or 137, a polypeptide (CDR-L2) includingan amino acid sequence of SEQ ID NO: 143 or 144, and a polypeptide(CDR-L3) including an amino acid sequence of SEQ ID NO: 149 or to 150,or a light chain variable region including the at least one light chaincomplementarity determining region;

a combination of the at least one heavy chain complementaritydetermining region and the at least one light chain complementaritydetermining region; or

a combination of the heavy chain variable region and the light chainvariable region.

In the anti-Ang2 antibody or an antigen-binding fragment thereof, theheavy chain variable region may include an amino acid sequence of SEQ IDNO: 162 or 163, and the light chain variable region may include an aminoacid sequence of SEQ ID NO: 170 or 171. Accordingly, the anti-Ang2antibody or an antigen-binding fragment thereof may include a heavychain variable region including an amino acid sequence of SEQ ID NO: 162or 163; a light chain variable region including an amino acid sequenceof SEQ ID NO: 170 or 171; or a combination of the heavy chain variableregion and the light chain variable region.

The anti-c-Met antibody may recognize a specific region of c-Met, e.g.,a specific region in the SEMA domain, as an epitope. It may be anyantibody or antigen-binding fragment that acts on c-Met to induce c-Metintracellular internalization and degradation.

c-Met, a receptor for hepatocyte growth factor (HGF), may be dividedinto three portions: extracellular, transmembrane, and intracellular.The extracellular portion is composed of an α-subunit and a β-subunitwhich are linked to each other through a disulfide bond, and includes aSEMA domain responsible for binding HGF, a plexin-semaphorins-integrinidentity/homology domain (PSI domain) and an immunoglobulin-like foldshared by plexins and transcriptional factors domain (IPT domain). TheSEMA domain of c-Met protein may have the amino acid sequence of SEQ IDNO: 79, and is an extracellular domain that functions to bind HGF. Aspecific region of the SEMA domain, that is, a region having the aminoacid sequence of SEQ ID NO: 71, which corresponds to a range from aminoacid residues 106 to 124 of the amino acid sequence of the SEMA domain(SEQ ID NO: 79), is a loop region between the second and the third betapropellers within the epitopes of the SEMA domain. This region acts asan epitope for the anti-c-Met antibody provided in the presentinvention.

The term “epitope,” as used herein, refers to an antigenic determinant,a part of an antigen recognized by an antibody. In one embodiment, theepitope may be a region including 5 or more contiguous (consecutive onprimary, secondary (two-dimensional), or tertiary (three-dimensional)structure) amino acid residues within the SEMA domain (SEQ ID NO: 79) ofc-Met protein, for instance, 5 to 19 contiguous amino acid residueswithin the amino acid sequence of SEQ ID NO: 71. For example, theepitope may be a polypeptide having 5 to 19 contiguous amino acidsselected from among partial combinations of the amino acid sequence ofSEQ ID NO: 71, wherein the polypeptide includes the amino sequence ofSEQ ID NO: 73 (EEPSQ) serving as an essential element for the epitope.For example, the epitope may be a polypeptide including, consistingessentially of, or consisting of the amino acid sequence of SEQ ID NO:71, SEQ ID NO: 72, or SEQ ID NO: 73.

The epitope having the amino acid sequence of SEQ ID NO: 72 correspondsto the outermost part of the loop between the second and third betapropellers within the SEMA domain of a c-Met protein. The epitope havingthe amino acid sequence of SEQ ID NO: 73 is a site to which the antibodyor antigen-binding fragment according to one embodiment mostspecifically binds.

Thus, the anti-c-Met antibody may specifically bind to an epitope whichincludes 5 to 19 contiguous amino acids selected from among partialcombinations of the amino acid sequence of SEQ ID NO: 71, including SEQID NO: 73 as an essential element. For example, the anti-c-Met antibodymay specifically bind to an epitope including the amino acid sequence ofSEQ ID NO: 71, SEQ ID NO: 72, or SEQ ID NO: 73.

In one embodiment, the anti-c-Met antibody or an antigen-bindingfragment thereof may include:

at least one heavy chain complementarity determining region (CDR)selected from the group consisting of (a) a CDR-H1 including the aminoacid sequence of SEQ ID NO: 4; (b) a

CDR-H2 including the amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 2,or an amino acid sequence including 8-19 consecutive amino acids withinSEQ ID NO: 2 including amino acid residues from the 3^(rd) to 10^(th)positions of SEQ ID NO: 2; and (c) a CDR-H3 including the amino acidsequence of SEQ ID NO: 6, SEQ ID NO: 85, or an amino acid sequenceincluding 6-13 consecutive amino acids within SEQ ID NO: 85 includingamino acid residues from the 1^(st) to 6^(th) positions of SEQ ID NO:85, or a heavy chain variable region including the at least one heavychain complementarity determining region;

at least one light chain complementarity determining region (CDR)selected from the group consisting of (a) a CDR-L1 including the aminoacid sequence of SEQ ID NO: 7, (b) a CDR-L2 including the amino acidsequence of SEQ ID NO: 8, and (c) a CDR-L3 including the amino acidsequence of SEQ ID NO: 9, SEQ ID NO: 15, SEQ ID NO: 86, or an amino acidsequence including 9-17 consecutive amino acids within SEQ ID NO: 89including amino acid residues from the 1^(st) to 9^(th) positions of SEQID NO: 89, or a light chain variable region including the at least onelight chain complementarity determining region;

a combination of the at least one heavy chain complementaritydetermining region and at least one light chain complementaritydetermining region; or

a combination of the heavy chain variable region and the light chainvariable region.

Herein, the amino acid sequences of SEQ ID NOS: 4 to 9 are respectivelyrepresented by following Formulas I to VI, below:

Formula I (SEQ ID NO: 4) Xaa₁-Xaa₂-Tyr-Tyr-Met-Ser,

wherein Xaa₁ is absent or Pro or Ser, and Xaa₂ is Glu or Asp,

Formula II (SEQ ID NO: 5) Arg-Asn-Xaa₃-Xaa₄-Asn-Gly-Xaa₅-Thr,

wherein Xaa₃ is Asn or Lys, Xaa₄ is Ala or Val, and Xaa₅ is Asn or Thr,

Formula III (SEQ ID NO: 6) Asp-Asn-Trp-Leu-Xaa₆-Tyr,

wherein Xaa₆ is Ser or Thr,

Formula IV (SEQ ID NO: 7) Lys-Ser-Ser-Xaa₇-Ser-Leu-Leu-Ala-Xaa₈-Gly-Asn-Xaa₉-Xaa₁₀-Asn-Tyr-Leu-Ala

wherein Xaa₇ is His, Arg, Gln, or Lys, Xaa₈ is Ser or Trp, Xaa₉ is Hisor Gln, and Xaa₁₀ is Lys or Asn,

Formula V (SEQ ID NO: 8) Trp-Xaa₁₁-Ser-Xaa₁₂-Arg-Val-Xaa₁₃

wherein Xaa₁₁ is Ala or Gly, Xaa₁₂ is Thr or Lys, and Xaa₁₃ is Ser orPro, and

Formula VI (SEQ ID NO: 9) Xaa₁₄-Gln-Ser-Tyr-Ser-Xaa₁₅-Pro-Xaa₁₆-Thr

wherein Xaa₁₄ is Gly, Ala, or Gln, Xaa₁₅ is Arg, His, Ser, Ala, Gly, orLys, and Xaa₁₆ is Leu, Tyr, Phe, or Met.

In one embodiment, the CDR-H1 may include an amino acid sequenceselected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 22, SEQID NO: 23, and SEQ ID NO: 24. The CDR-H2 may include an amino acidsequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO:25, and SEQ ID NO: 26. The CDR-H3 may include an amino acid sequenceselected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 27, SEQID NO: 28, and SEQ ID NO: 85.

The CDR-L1 may include an amino acid sequence selected from the groupconsisting of SEQ ID NO: 10, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO:31, SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO: 106. The CDR-L2 mayinclude an amino acid sequence selected from the group consisting of SEQID NO: 11, SEQ ID NO: 34, SEQ ID NO: 35, and SEQ ID NO: 36. The CDR-L3may include an amino acid sequence selected from the group consisting ofSEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO:16, SEQ ID NO: 37, SEQ ID NO: 86, and SEQ ID NO: 89.

In another embodiment, the antibody or antigen-binding fragment mayinclude a heavy variable region including a polypeptide (CDR-H1)including an amino acid sequence selected from the group consisting ofSEQ ID NO: 1, SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24, apolypeptide (CDR-H2) including an amino acid sequence selected from thegroup consisting of SEQ ID NO: 2, SEQ ID NO: 25, and SEQ ID NO: 26, anda polypeptide (CDR-H3) including an amino acid sequence selected fromthe group consisting of SEQ ID NO: SEQ ID NO: 3, SEQ ID NO: 27, SEQ IDNO: 28, and SEQ ID NO: 85; and a light variable region including apolypeptide (CDR-L1) including an amino acid sequence selected from thegroup consisting of SEQ ID NO: 10, SEQ ID NO: 29, SEQ ID NO: 30, SEQ IDNO: 31, SEQ ID NO: 32, SEQ ID NO: 33, and SEQ ID NO: 106, a polypeptide(CDR-L2) including an amino acid sequence selected from the groupconsisting of SEQ ID NO: 11, SEQ ID NO: 34, SEQ ID NO: 35, and SEQ IDNO: 36, and a polypeptide (CDR-L3) including an amino acid sequenceselected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 13, SEQID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 37, SEQ ID NO: 86,and SEQ ID NO: 89.

In one embodiment, the anti-c-Met antibody or an antigen-bindingfragment thereof may be modified by the deletion, insertion, addition,or substitution of at least one amino acid residue on the amino acidsequence of the hinge region so that it exhibit enhanced antigen-bindingefficiency. For example, the antibody may include a hinge region havingthe amino acid sequence of SEQ ID NO: 100 (U7-HC6), SEQ ID NO: 101(U6-HC7), SEQ ID NO: 102 (U3-HC9), SEQ ID NO: 103 (U6-HC8), or SEQ IDNO: 104 (U8-HC5), or a hinge region having the amino acid sequence ofSEQ ID NO: 105 (non-modified human hinge). In particular, the hingeregion includes the amino acid sequence of SEQ ID NO: 100 or SEQ ID NO:101.

In one embodiment, the anti-c-Met antibody or antigen-binding fragmentmay have a heavy chain variable region including the amino acid sequenceof SEQ ID NO: 17, SEQ ID NO: 74, SEQ ID NO: 87, SEQ ID NO: 90, SEQ IDNO: 91, SEQ ID NO: 92, SEQ ID NO: 93, or SEQ ID NO: 94; a light chainvariable region including the amino acid sequence of SEQ ID NO: 18, SEQID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 75, SEQ ID NO: 88,SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO:99, or SEQ ID NO: 107; or a combination of the heavy chain variableregion and the light chain variable region.

In one embodiment, the anti-c-Met antibody may be a monoclonal antibody.The monoclonal antibody may be produced by the hybridoma cell linedeposited with the Korean Cell Line Research Foundation, aninternational depository authority located at Yungun-Dong, Jongno-Gu,Seoul, Korea, on Oct. 6, 2009, under Accession No. KCLRF-BP-00220, whichbinds specifically to the extracellular region of c-Met protein (referto Korean Patent Publication No. 2011-0047698, the disclosure of whichis incorporated in its entirety herein by reference). The anti-c-Metantibody may include all the antibodies defined in Korean PatentPublication No. 2011-0047698.

In the anti-c-Met antibody or an antigen-binding fragment thereof andthe anti-Ang2 antibody or an antigen-binding fragment thereof, theportion of the light chain and the heavy chain portion excluding theCDRs, the light chain variable region, and the heavy chain variableregion as defined above, that is the light chain constant region and theheavy chain constant region, may be those from any subtype ofimmunoglobulin (e.g., IgA, IgD, IgE, IgG (IgG1, IgG2, IgG3, IgG4), IgM,etc.).

By way of further example, the anti-c-Met antibody or the antibodyfragment may include:

a heavy chain including the amino acid sequence selected from the groupconsisting of the amino acid sequence of SEQ ID NO: 62 (wherein theamino acid sequence from amino acid residues from the 1^(st) to 17^(th)positions is a signal peptide), or the amino acid sequence from the18^(th) to 462^(nd) positions of SEQ ID NO: 62, the amino acid sequenceof SEQ ID NO: 64 (wherein the amino acid sequence from the 1^(st) to17^(th) positions is a signal peptide), the amino acid sequence from the18^(th) to 461^(st) positions of SEQ ID NO: 64, the amino acid sequenceof SEQ ID NO: 66 (wherein the amino acid sequence from the 1^(st) to17^(th) positions is a signal peptide), and the amino acid sequence fromthe 18^(th) to 460^(th) positions of SEQ ID NO: 66; and

a light chain including the amino acid sequence selected from the groupconsisting of the amino acid sequence of SEQ ID NO: 68 (wherein theamino acid sequence from the 1^(st) to 20^(th) positions is a signalpeptide), the amino acid sequence from the 21^(st) to 240^(th) positionsof SEQ ID NO: 68, the amino acid sequence of SEQ ID NO: 70 (wherein theamino acid sequence from the 1^(st) to 20^(th) positions is a signalpeptide), the amino acid sequence from the 21^(st) to 240^(th) positionsof SEQ ID NO: 70, and the amino acid sequence of SEQ ID NO: 108.

For example, the anti-c-Met antibody may be selected from the groupconsisting of:

an antibody including a heavy chain including the amino acid sequence ofSEQ ID NO: 62 or the amino acid sequence from the 18^(th) to 462^(nd)positions of SEQ ID NO: 62 and a light chain including the amino acidsequence of SEQ ID NO: 68 or the amino acid sequence from the 21^(st) to240^(th) positions of SEQ ID NO: 68;

an antibody including a heavy chain including the amino acid sequence ofSEQ ID NO: 64 or the amino acid sequence from the 18^(th) to 461^(st)positions of SEQ ID NO: 64 and a light chain including the amino acidsequence of SEQ ID NO: 68 or the amino acid sequence from the 21^(st) to240^(th) positions of SEQ ID NO: 68;

an antibody including a heavy chain including the amino acid sequence ofSEQ ID NO: 66 or the amino acid sequence from the 18^(th) to 460^(th)positions of SEQ ID NO: 66 and a light chain including the amino acidsequence of SEQ ID NO: 68 or the amino acid sequence from the 21^(st) to240^(th) positions of SEQ ID NO: 68;

an antibody including a heavy chain including the amino acid sequence ofSEQ ID NO: 62 or the amino acid sequence from the 18^(th) to 462^(nd)positions of SEQ ID NO: 62 and a light chain including the amino acidsequence of SEQ ID NO: 70 or the amino acid sequence from the 21^(st) to240^(th) positions of SEQ ID NO: 70;

an antibody including a heavy chain including the amino acid sequence ofSEQ ID NO: 64 or the amino acid sequence from the 18^(th) to 461^(st)positions of SEQ ID NO: 64 and a light chain including the amino acidsequence of SEQ ID NO: 70 or the amino acid sequence from the 21^(st) to240^(th) positions of SEQ ID NO: 70;

an antibody including a heavy chain including the amino acid sequence ofSEQ ID NO: 66 or the amino acid sequence from the 18^(th) to 460^(th)positions of SEQ ID NO: 66 and a light chain including the amino acidsequence of SEQ ID NO: 70 or the amino acid sequence from the 21^(st) to240^(th) positions of SEQ ID NO: 70;

an antibody including a heavy chain including the amino acid sequence ofSEQ ID NO: 62 or the amino acid sequence from the 18^(th) to 462^(nd)positions of SEQ ID NO: 62 and a light chain including the amino acidsequence of SEQ ID NO: 108;

an antibody including a heavy chain including the amino acid sequence ofSEQ ID NO: 64 or the amino acid sequence from the 18^(th) to 461^(st)positions of SEQ ID NO: 64 and a light chain including the amino acidsequence of SEQ ID NO: 108; and

an antibody including a heavy chain including the amino acid sequence ofSEQ ID NO: 66 or the amino acid sequence from the 18^(th) to 460^(th)positions of SEQ ID NO: 66 and a light chain including the amino acidsequence of SEQ ID NO: 108.

The polypeptide of SEQ ID NO: 70 is a light chain including human kappa(κ) constant region, and the polypeptide with the amino acid sequence ofSEQ ID NO: 68 is a polypeptide obtained by replacing histidine atposition 62 (corresponding to position 36 of SEQ ID NO: 68 according tokabat numbering) of the polypeptide with the amino acid sequence of SEQID NO: 70 with tyrosine. The production yield of the antibodies may beincreased by the replacement. The polypeptide with the amino acidsequence of SEQ ID NO: 108 is a polypeptide obtained by replacing serineat position 32 (position 27e according to kabat numbering in the aminoacid sequence from amino acid residues 21 to 240 of SEQ ID NO: 68;positioned within CDR-L1) with tryptophan. By such replacement,antibodies and antibody fragments including such sequences exhibitsincreased activities, such as c-Met biding affinity, c-Met degradationactivity, Akt phosphorylation inhibition, and the like.

In another embodiment, the anti-c-Met antibody may include a light chaincomplementarity determining region including the amino acid sequence ofSEQ ID NO: 106, a light chain variable region including the amino acidsequence of SEQ ID NO: 107, or a light chain including the amino acidsequence of SEQ ID NO: 108.

In another embodiment, the anti-c-Met antibody may include a heavy chainincluding the amino acid sequence of SEQ ID NO: 66 or the amino acidsequence from the 18^(th) to 460^(th) positions of SEQ ID NO: 66 and alight chain including the amino acid sequence of SEQ ID NO: 68 or theamino acid sequence from the 21^(st) to 240^(th) positions of SEQ ID NO:68.

The following descriptions may be applied to both the anti-c-Metantibodies or fragments thereof and the anti-Ang2 antibodies orfragments thereof described herein.

Animal-derived antibodies produced by immunizing non-immune animals witha desired antigen generally invoke immunogenicity when injected tohumans for the purpose of medical treatment, and thus chimericantibodies have been developed to inhibit such immunogenicity. Chimericantibodies are prepared by replacing constant regions of animal-derivedantibodies that cause an anti-isotype response with constant regions ofhuman antibodies by genetic engineering. Chimeric antibodies areconsiderably improved in an anti-isotype response compared toanimal-derived antibodies, but animal-derived amino acids still havevariable regions, so that chimeric antibodies have side effects withrespect to a potential anti-idiotype response. Humanized antibodies havebeen developed to reduce such side effects. Humanized antibodies areproduced by grafting complementarity determining regions (CDR) whichserve an important role in antigen binding in variable regions ofchimeric antibodies into a human antibody framework.

The most important thing in CDR grafting to produce humanized antibodiesis choosing the optimized human antibodies for accepting CDRs ofanimal-derived antibodies. Antibody databases, analysis of a crystalstructure, and technology for molecule modeling are used. However, evenwhen the CDRs of animal-derived antibodies are grafted to the mostoptimized human antibody framework, amino acids positioned in aframework of the animal-derived CDRs affecting antigen binding arepresent. Therefore, in many cases, antigen binding affinity is notmaintained, and thus application of additional antibody engineeringtechnology for recovering the antigen binding affinity is necessary.

The antibodies, e.g., the anti c-Met antibodies, the anti-Ang2antibodies, and the anti-c-Met/anti-Ang2 bispecific antibody may beanimal antibodies (e.g., mouse-derived antibodies), chimeric antibodies(e.g., mouse-human chimeric antibodies), humanized antibodies, or humanantibodies. The antibodies or antigen-binding fragments thereof may beisolated from a living body or non-naturally occurring. The antibodiesor antigen-binding fragments thereof may be synthetic or recombinant.The antibodies or antigen-binding fragments thereof may be monoclonal.

An intact antibody includes two full-length light chains and twofull-length heavy chains, in which each light chain is linked to a heavychain by disulfide bonds. The antibody has a heavy chain constant regionand a light chain constant region. The heavy chain constant region is ofa gamma (γ), mu (μ), alpha (α), delta (δ), or epsilon (ε) type, whichmay be further categorized as gamma 1 (γ1), gamma 2 (γ2), gamma 3 (γ3),gamma 4 (γ4), alpha 1 (α1), or alpha 2 (α2). The light chain constantregion is of either a kappa (κ) or lambda (λ) type.

As used herein, the term “heavy chain” refers to full-length heavychain, and fragments thereof, including a variable region V_(H) thatincludes amino acid sequences sufficient to provide specificity toantigens, and three constant regions, C_(II1), C_(II2), and C_(II3), anda hinge. The term “light chain” refers to a full-length light chain andfragments thereof, including a variable region V_(L) that includes aminoacid sequences sufficient to provide specificity to antigens, and aconstant region C_(L).

The term “complementarity determining region (CDR)” refers to an aminoacid sequence found in a hyper variable region of a heavy chain or alight chain of immunoglobulin. The heavy and light chains mayrespectively include three CDRs (CDRH1, CDRH2, and CDRH3; and CDRL1,CDRL2, and CDRL3). The CDR may provide contact residues that play animportant role in the binding of antibodies to antigens or epitopes. Theterms “specifically binding” and “specifically recognized” are wellknown to one of ordinary skill in the art, and indicate that an antibodyand an antigen specifically interact with each other to lead to animmunological activity.

The term “antigen-binding fragment” used herein refers to fragments ofan intact immunoglobulin including portions of a polypeptide includingantigen-binding regions having the ability to specifically bind to theantigen. In a particular embodiment, the antigen-binding fragment may bescFv, (scFv)₂, scFvFc, Fab, Fab′, or F(ab′)₂, but is not limitedthereto.

Among the antigen-binding fragments, Fab that includes light chain andheavy chain variable regions, a light chain constant region, and a firstheavy chain constant region C_(H1), has one antigen-binding site.

The Fab′ fragment is different from the Fab fragment, in that Fab′includes a hinge region with at least one cysteine residue at theC-terminal of C_(H1).

The F(ab′)₂ antibody is formed through disulfide bridging of thecysteine residues in the hinge region of the Fab′ fragment.

Fv is the smallest antibody fragment with only a heavy chain variableregion and a light chain variable region. Recombination techniques ofgenerating the Fv fragment are widely known in the art.

Two-chain Fv includes a heavy chain variable region and a light chainregion which are linked by a non-covalent bond. Single-chain Fvgenerally includes a heavy chain variable region and a light chainvariable region which are linked by a covalent bond via a peptide linkeror linked at the C-terminals to have a dimer structure like thetwo-chain Fv. The peptide linker may be the same as described in theabove, for example, those including the amino acid length of about 1 toabout 100, about 2 to about 50, particularly about 5 to about 25, andany kinds of amino acids may be included without any restrictions.

The antigen-binding fragments may be attainable using protease (forexample, the Fab fragment may be obtained by restricted cleavage of awhole antibody with papain, and the F(ab′)₂ fragment may be obtained bycleavage with pepsin), or may be prepared by using a geneticrecombination technique.

The term “hinge region,” as used herein, refers to a region betweenC_(H1) and C_(H2) domains within the heavy chain of an antibody whichfunctions to provide flexibility for the antigen-binding site.

When an animal antibody undergoes a chimerization process, the IgG1hinge of animal origin is replaced with a human IgG1 hinge or IgG2 hingewhile the disulfide bridges between two heavy chains are reduced fromthree to two in number. In addition, an animal-derived IgG1 hinge isshorter than a human IgG1 hinge. Accordingly, the rigidity of the hingeis changed. Thus, a modification of the hinge region may bring about animprovement in the antigen binding efficiency of the humanized antibody.The modification of the hinge region through amino acid deletion,addition, or substitution is well-known to those skilled in the art.

The anti-Ang2 antibody may be a monoclonal antibody. The monoclonalantibody may be prepared by methods well known in the art. For example,it may be prepared using a phage display technique.

Meanwhile, individual monoclonal antibodies may be screened using atypical ELISA (Enzyme-Linked ImmunoSorbent Assay) format, based on thebinding potential with Ang2 or c-Met. Inhibitory activities can beverified through functional analysis such as competitive ELISA forverifying the molecular interaction of binding assemblies or functionalanalysis such as a cell-based assay. Then, with regard to monoclonalantibody members selected on the basis of their strong inhibitoryactivities, their affinities (Kd values) to Ang2 or c-Met may be eachverified.

In an embodiment, the anti-c-Met/anti-Ang2 bispecific antibody mayinclude an anti-c-Met antibody or an antigen-binding fragment thereof,and an anti-Ang2 antibody or an antigen-binding fragment thereof,wherein the anti-Ang2 antibody or an antigen-binding fragment thereof islinked to C-terminus or N-terminus, e.g., C-terminus, of the anti-c-Metantibody or an antigen-binding fragment thereof.

As used herein, the term “bispecific antibody” refers to a fusionpolypeptide having an antibody or antibody-like structure, whichcomprises a first antibody specifically recognizing and/or binding to afirst antigen or an antigen-binding fragment of the first antibody, anda second antibody specifically recognizing and/or binding to a secondantigen (different from the first antigen) or an antigen-bindingfragment of the second antibody, wherein the second antigen or anantigen-binding fragment thereof is linked to the C-terminus of theheavy chain or the light chain of the first antibody or anantigen-binding fragment thereof directly or via a proper linker.

In the anti-c-Met/anti-Ang2 bispecific antibody, in order to fullyperform the anti-c-Met antibody's activity to mediate intracellularmigration and degradation of c-Met proteins, it may be advantageous thatthe anti-c-Met antibody has its own intact antibody structure. Inaddition, in case of the anti-Ang2 antibody, its specific recognitionand binding to Ang2 is important, and thus it will be fine that just anantigen-binding fragment recognizing Ang2 is included in the bispecificantibody. Therefore, the anti-c-Met/anti-Ang2 bispecific antibody maycomprise a complete anti-c-Met antibody (e.g., IgG type antibody;comprising two heavy chains and two light chains) and an antigen bindingfragment (e.g., scFv or scFv-Fc) of the anti-Ang2 antibody linked to theC terminus of the anti-c-Met antibody, but not be limited thereto.

In the anti-c-Met/anti-Ang2 bispecific antibody, the anti-c-Met antibodyor the antigen binding fragment thereof, and the anti-Ang2 antibody orthe antigen binding fragment thereof, may be linked via a peptide linkeror without it. Furthermore, a heavy chain portion and a light chainportion within the antigen binding fragment, for example, a heavy chainvariable region and a light chain variable region within the scFvfragment, may be linked via a peptide linker or without it. The peptidelinker which links the anti-c-Met antibody or the antigen bindingfragment thereof, and the anti-EGFR antibody or the antigen bindingfragment thereof, and the peptide linker which links the heavy chainportion and the light chain portion within the antigen binding fragmentmay be identical or different. The peptide linker may be about 1 toabout 100, particularly about 2 to about 50, amino acids in length andinclude any kinds of amino acids. The peptide linker may include forexample, Gly, Asn and/or Ser residues, and also include neutral aminoacids such as Thr and/or Ala. Amino acid sequences suitable for thepeptide linker are known in the relevant art. The length of the peptidelinker may be determined within such a limit that the functions of thefusion protein (bispecific antibody) will not be affected. For instance,the peptide linker may be formed by including a total of about 1 toabout 100, about 2 to about 50, or about 5 to about 25 of one or moreamino acids selected from the group consisting of Gly, Asn, Ser, Thr,and Ala. In one embodiment, the peptide linker may be represented as(GGGGS)n, wherein n is a repeat number of (GGGGS), which is an integerof about 1 to about 10, particularly an integer of about 2 to about 5.

In a particular embodiment, the anti-c-Met/anti-Ang2 bispecific antibodymay include an anti-c-Met antibody, and an scFv, (scFv)₂, Fab, Fab′ orF(ab′)₂, for example, scFv, of an anti-Ang2 antibody linked to the Cterminus of the anti-c-Met antibody. For instance, scFv, (scFv)₂, Fab,Fab′ or F(ab′)₂ of the anti-EGFR antibody may include a heavy chainvariable region including the amino acid sequence selected from SEQ IDNO: 156 to SEQ ID NO: 163, and a light chain variable region includingthe amino acid sequence selected from SEQ ID NO: 164 to SEQ ID NO: 171.

Hence, in a particular embodiment, the anti-c-Met/anti-Ang2 bispecificantibody may include an anti-c-Met antibody, and an scFv, (scFv)₂, Fab,Fab′ or F(ab′)₂ of an anti-Ang2 antibody including a heavy chainvariable region including the amino acid sequence selected from SEQ IDNO: 156 to SEQ ID NO: 163, and a light chain variable region the aminoacid sequence selected from SEQ ID NO: 164 to SEQ ID NO: 171, which islinked to the C terminal of the anti-c-Met antibody.

In an embodiment, the heavy chain of the anti-c-Met/anti-Ang2 bispecificantibody may include the amino acid sequence of SEQ ID NO: 189 (whereinthe amino acid sequence from the 1^(st) to 17^(th) positions is a signalpeptide) or the amino acid sequence from the 18^(th) to 738^(th)positions of the amino acid sequence of SEQ ID NO: 189. The light chainof the anti-c-Met/anti-Ang2 bispecific antibody may be the same withthat of the above described anti-c-Met antibody, and for example, lightchain may include an amino acid sequence selected from the groupconsisting of the amino acid sequence of SEQ ID NO: 68 (wherein theamino acid sequence from the 1^(st) to 20^(th) positions is a signalpeptide), the amino acid sequence from the 21^(st) to 240^(th) positionsof the amino acid sequence of SEQ ID NO: 68, the amino acid sequence ofSEQ ID NO: 70 (wherein the amino acid sequence from the 1^(st) to20^(th) positions is a signal peptide), the amino acid sequence from the21″ to 240^(th) positions of the amino acid sequence of SEQ ID NO: 70,and the amino acid sequence of SEQ ID NO: 108.

Due to internalization and degradation activities of the anti-c-Metantibody, the anti-c-Met/anti-Ang2 bispecific antibody is capable of notonly inhibiting the activities of c-Met and Ang2, but also decreasingthe total amount of c-Met and Ang2 by degrading them, thereby leading tomore fundamental blocking of activity of the c-Met and Ang2. Therefore,the anti-c-Met/anti-Ang2 bispecific antibody can exhibit therapeuticeffects even when it is applied to a subject who has a resistanceagainst a preexisting Ang2-targeting drug, such as an anti-Ang2, or ananti-c-Met antibody.

In another embodiment, provided is a pharmaceutical compositionincluding the anti-c-Met/anti-Ang2 bispecific antibody.

Another embodiment provides a pharmaceutical composition for preventingand/or treating a disease associated with Ang2/Tie2 signal transductionsystem and/or HGF/c-Met signal transduction system, wherein thecomposition includes the anti-c-Met/anti-Ang2 bispecific antibody as anactive ingredient.

Another embodiment provides a method of preventing and/or treating adisease associated with Ang2/Tie2 signal transduction system and/orHGF/c-Met signal transduction system, including administering theanti-c-Met/anti-Ang2 bispecific antibody to a subject in need ofpreventing and/or treating a disease associated with Ang2/Tie2 signaltransduction system and/or HGF/c-Met signal transduction system. Theanti-c-Met/anti-Ang2 bispecific antibody may be administered in apharmaceutically effective amount for preventing and/or treating adisease associated with Ang2/Tie2 signal transduction system and/orHGF/c-Met signal transduction system. The method may further include astep of identifying the subject in need of preventing and/or treating adisease associated with Ang2/Tie2 signal transduction system and/orHGF/c-Met signal transduction system, before the step of administering.

The disease associated with Ang2/Tie2 signal transduction system and/orHGF/c-Met signal transduction system may be a disease related toincrease of angiogenesis and/or increase of vascular permeability and/orover-expression of Ang2 and/or c-Met, and for example, the disease maybe at least one selected from the group consisting of cancers; cancermetastasis; eye diseases such as retinopathy of prematurity, maculardegeneration (e.g., age-related macular degeneration), diabeticretinopathy, glaucoma (e.g., neovascular glaucoma) etc.; inflammatorydiseases (infection) such as psoriasis, rheumatoid arthritis, pneumonia,chronic inflammation, etc.; cardiovascular diseases such ashypertension, arteriosclerosis, etc.; renal diseases; sepsis; malaria;asthma; edema; hereditary hemorrhagic telangiectasia (HHT); and thelike.

Another embodiment may provide a pharmaceutical composition forpresenting and/or treating a cancer, wherein the composition includesthe anti-c-Met/anti-Ang2 bispecific antibody as an active ingredient.

Another embodiment provides a method of preventing and/or treating acancer, including administering the anti-c-Met/anti-Ang2 bispecificantibody to a subject in need of preventing and/or treating a cancer.The anti-c-Met/anti-Ang2 bispecific antibody may be administered in apharmaceutically effective amount for preventing and/or treating acancer. The method may further include a step of identifying the subjectin need of preventing and/or treating cancer, before the step ofadministering.

The prevention and/or treatment of a cancer may refer to preventionand/or treatment of a cancer and/or cancer metastasis.

The cancer may be a solid cancer or a blood cancer, and it may be atleast one selected from the group consisting of squamous cell carcinoma,small-cell lung cancer, non-small-cell lung cancer, adenocarcinoma ofthe lung, squamous cell carcinoma of the lung, peritoneal carcinoma,skin cancer, melanoma in the skin or eyeball, rectal cancer, cancer nearthe anus, esophagus cancer, small intestinal tumor, endocrine glandcancer, parathyroid cancer, adrenal cancer, soft-tissue sarcoma,urethral cancer, chronic or acute leukemia, lymphocytic lymphoma,hepatocellular cancer, gastrointestinal cancer, gastric cancer,pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, livercancer, bladder cancer, hepatoma, breast cancer, colon cancer, largeintestine cancer, endometrial carcinoma or uterine carcinoma, salivarygland tumor, kidney cancer, prostate cancer, vulvar cancer, thyroidcancer, head or neck cancer, brain cancer, osteosarcoma, and the like,but not limited thereto. In particular, the cancer may be one having aresistance against a preexisting anticancer drug, such as an antagonistto c-Met or an antagonist to Ang2. The cancer may be a primary cancer ora metastatic cancer.

In the pharmaceutical composition or method, the anti-c-Met/anti-Ang2bispecific antibody may be administered together with one or moreadditives selected from the group consisting of pharmaceuticallyacceptable carriers, diluents, excipients, and the like.

The pharmaceutically acceptable carrier may be any one commonly used inthe formulation of drugs, which may be one or more selected from thegroup consisting of lactose, dextrose, sucrose, sorbitol, mannitol,starch, gum acacia, calcium phosphate, alginates, gelatin, calciumsilicate, micro-crystalline cellulose, polyvinylpyrrolidone, cellulose,water, syrup, methyl cellulose, methylhydroxy benzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil, but is not limitedthereto. The pharmaceutical composition may further include one or moreselected from the group consisting of a diluent, an excipient, alubricant, a wetting agent, a sweetener, a flavor enhancer, anemulsifying agent, a suspension agent, and a preservative.

The pharmaceutical composition, the anti-Ang2 antibody or anantigen-binding fragment thereof, or the anti-c-Met/anti-Ang2 bispecificantibody may be administered orally or parenterally. The parenteraladministration may include intravenous injection, subcutaneousinjection, muscular injection, intraperitoneal injection, endothelialadministration, local administration, intranasal administration,intrapulmonary administration, and rectal administration. Since oraladministration leads to digestion of proteins or peptides, an activeingredient in the compositions for oral administration must be coated orformulated to prevent digestion in stomach. In addition, the compositionmay be administered using an optional device that enables an activesubstance to be delivered to target cells.

The suitable dose of the pharmaceutical composition, the anti-Ang2antibody or an antigen-binding fragment thereof, or theanti-c-Met/anti-Ang2 bispecific antibody may be prescribed in a varietyof ways, depending on factors such as formulation methods,administration methods, age of subjects, body weight, gender, pathologicconditions, diets, administration time, administration interval,administration route, excretion speed, and reaction sensitivity. Forexample, a daily dosage of the composition the anti-Ang2 antibody or anantigen-binding fragment thereof, or the anti-c-Met/anti-Ang2 bispecificantibody may be within the range of about 0.001 to about 1000 mg/kg(e.g., about 0.001 mg/kg, about 0.01 mg/kg, about 1 mg/kg, or about 10mg/kg), particularly about 0.01 to about 100 mg/kg (e.g., about 0.01mg/kg, about 0.1 mg/kg, about 0.5 mg/kg, about 1 mg/kg, or about 5mg/kg), and more particularly about 0.1 to about 50 mg/kg (e.g., about0.1 mg/kg, about 1 mg/kg, about 10 mg/kg, or about 50 mg/kg), but is notlimited thereto. The daily dosage may be formulated into a singleformulation in a unit dosage form or formulated in suitably divideddosage forms, or it may be manufactured to be contained in a multipledosage container. The term “pharmaceutically effective amount” as usedherein refers to a content or dose of an active ingredient capable ofshowing desirable pharmacological effects and it may be determined in avariety of ways, depending on factors such as formulation methods,administration methods, age of subjects, body weight, gender, pathologicconditions, diets, administration time, administration interval,administration route, excretion speed, and reaction sensitivity.

The pharmaceutical composition or the anti-c-Met/anti-Ang2 bispecificantibody may be formulated with a pharmaceutically acceptable carrierand/or excipient into a unit or a multiple dosage form by a methodeasily carried out by a skilled person in the pertinent art. The dosageform may be a solution in oil or an aqueous medium, a suspension, syrup,an emulsifying solution, an extract, powder, granules, a tablet, or acapsule, and may further include a dispersing or a stabilizing agent.

In addition, the pharmaceutical composition or the anti-c-Met/anti-Ang2bispecific antibody may be administered as an individual drug, ortogether with other drugs, and may be administered sequentially orsimultaneously with pre-existing drugs.

In particular, the pharmaceutical composition including an antibody oran antigen-binding fragment thereof may be formulated into animmunoliposome since it contains an antibody or an antigen-bindingfragment. A liposome containing an antibody may be prepared using anymethods widely known in the art. The immunoliposome may be a lipidcomposition including phosphatidylcholine, cholesterol, andpolyethyleneglycol-derivatized phosphatidylethanolamine, and may beprepared by a reverse phase evaporation method. For example, Fab′fragments of an antibody may be conjugated to the liposome through adisulfide-exchange reaction.

The subject to whom the pharmaceutical composition or theanti-c-Met/anti-Ang2 bispecific antibody is administered may be mammals,for example, primates such as humans and monkeys, or rodents such asrats and mice, or a cell or a tissue isolated from the mammal or aculture thereof, but are not be limited thereto. The subject may be acancer patient having resistance against pre-existing anticancer drugs,for example, antagonists against a cancer-related target (e.g., c-Met,Ang2, etc.).

Another embodiment provides a polynucleotide encoding theanti-c-Met/anti-Ang2 bispecific antibody. For example, thepolynucleotide may comprise or consist essentially of (i) apolynucleotide encoding a polypeptide comprising a heavy chain of ananti-c-Met antibody and an antigen-binding fragment (e.g., an scFvfragment) of an anti-Ang2 antibody linked to the C-terminus of the heavychain of an anti-c-Met antibody, (ii) a polynucleotide encoding apolypeptide comprising a light chain of an anti-c-Met antibody, or (iii)a combination of (i) and (ii).

Another embodiment provides a recombinant vector comprising (orcarrying) the polynucleotide. Another embodiment provides a recombinantcell comprising (or transfected with) the polynucleotide or therecombinant vector.

Another embodiment provides a method of preparing theanti-c-Met/anti-Ang2 bispecific antibody, comprising expressing apolynucleotide encoding the anti-c-Met/anti-Ang2 bispecific antibody ina cell. As described above, the polynucleotide may comprise or consistessentially of (i) a polynucleotide encoding a polypeptide comprising aheavy chain of an anti-c-Met antibody and an antigen-binding fragment(e.g., an scFv fragment) of an anti-Ang2 antibody linked to theC-terminus of the heavy chain of an anti-c-Met antibody, (ii) apolynucleotide encoding a polypeptide comprising a light chain of ananti-c-Met antibody, or (iii) a combination of (i) and (ii). In themethod, the nucleotides (i) and (ii) are carried into a cell together byone vector or separately by two vectors. Another embodiment provides amethod of preparing the anti-c-Met/anti-Ang2 bispecific antibody,comprising linking an anti-Ang2 antibody or an antigen-binding fragmentthereof to C-terminus or N-terminus of an ant-c-Met antibody or anantigen-binding fragment thereof. For example, the method may compriselinking an antigen-binding fragment (e.g., an scFv fragment) of ananti-Ang2 antibody to a c-terminus of an anti-c-Met antibody.

In the nucleotide, the vector, the cell, and the method, theanti-c-Met/anti-Ang2 bispecific antibody, the heavy chain of ananti-c-Met antibody, the light chain of an anti-c-Met antibody, and theantigen-binding fragment (e.g., an scFv fragment) of an anti-Ang2antibody are as described above.

The anti-c-Met/anti-Ang2 antibody possess both characteristics as aninhibitor against Ang2/Tie2 signal transduction and c-Met/HGF signaltransduction, and may exhibit the following effects of:

1. Increasing the therapeutic efficacy compared to administration ofAng2/Tie2 inhibitor alone, HGF/c-Met inhibitor alone, or a combinationthereof, thereby decreasing the administration amount,

2. being used in treatment of a metastatic cancer as well as a primarycancer, and

3. being applied to a disease associated with Ang2/Tie2 signaltransduction and HGF/c-Met signal transduction, other than cancers.

EXAMPLES

Hereafter, the present invention will be described in detail byexamples.

The following examples are intended merely to illustrate the inventionand are not construed to restrict the invention.

Reference Example 1 Construction of an Anti-c-Met Antibody

1.1. Production of “AbF46”, a Mouse Antibody to c-Met

1.1.1. Immunization of a Mouse

To obtain immunized mice necessary for the development of a hybridomacell line, each of five BALB/c mice (Japan SLC, Inc.), 4 to 6 weeks old,was intraperitoneally injected with a mixture of 100 μg of humanc-Met/Fc fusion protein (R&D Systems) and one volume of completeFreund's adjuvant. Two weeks after the injection, a secondintraperitoneal injection was conducted on the same mice with a mixtureof 50 μg of human c-Met/Fc protein and one volume of incomplete Freund'sadjuvant. One week after the second immunization, the immune responsewas finally boosted. Three days later, blood was taken from the tails ofthe mice and the sera were 1/1000 diluted in PBS and used to examine atiter of antibody to c-Met by ELISA. Mice found to have a sufficientantibody titer were selected for use in the cell fusion process.

1.1.2. Cell fusion and Production of a Hybridoma

Three days before cell fusion, BALB/c mice (Japan SLC, Inc.) wereimmunized with an intraperitoneal injection of a mixture of 50 μg ofhuman c-Met/Fc fusion protein and one volume of PBS. The immunized micewere anesthetized before excising the spleen from the left half of thebody. The spleen was meshed to separate splenocytes which were thensuspended in a culture medium (DMEM, GIBCO, Invitrogen). The cellsuspension was centrifuged to recover the cell layer. The splenocytesthus obtained (1×10⁸ cells) were mixed with myeloma cells (Sp2/0) (1×10⁸cells), followed by spinning to give a cell pellet. The cell pellet wasslowly suspended, treated with 45% polyethylene glycol (PEG) (1 mL) inDMEM for 1 min at 37° C., and supplemented with 1 mL of DMEM. To thecells was added 10 mL of DMEM over 10 min, after which incubation wasconducted in a water bath at 37° C. for 5 min. Then the cell volume wasadjusted to 50 mL before centrifugation. The cell pellet thus formed wasresuspended at a density of 1-2×10⁵ cells/mL in a selection medium (HATmedium) and 0.1 mL of the cell suspension was allocated to each well of96-well plates which were then incubated at 37° C. in a CO₂ incubator toestablish a hybridoma cell population.

1.1.3. Selection of Hybridoma Cells Producing Monoclonal Antibodies toc-Met Protein

From the hybridoma cell population established in Reference Example1.1.2, hybridoma cells which showed a specific response to c-Met proteinwere screened by ELISA using human c-Met/Fc fusion protein and human Fcprotein as antigens.

Human c-Met/Fc fusion protein was seeded in an amount of 50 μL (2μg/mL)/well to microtiter plates and allowed to adhere to the surface ofeach well. The antibody that remained unbound was removed by washing.For use in selecting the antibodies that do not bind c-Met but recognizeFc, human Fc protein was attached to the plate surface in the samemanner.

The hybridoma cell culture obtained in Reference Example 1.1.2 was addedin an amount of 50 μL to each well of the plates and incubated for 1hour. The cells remaining unreacted were washed out with a sufficientamount of Tris-buffered saline and Tween 20 (TBST). Goat anti-mouseIgG-horseradish peroxidase (HRP) was added to the plates and incubatedfor 1 hour at room temperature. The plates were washed with a sufficientamount of TBST, followed by reacting the peroxidase with a substrate(OPD). Absorbance at 450 nm was measured on an ELISA reader.

Hybridoma cell lines which secrete antibodies that specifically andstrongly bind to human c-Met but not human Fc were selected repeatedly.From the hybridoma cell lines obtained by repeated selection, a singleclone producing a monoclonal antibody was finally separated by limitingdilution. The single clone of the hybridoma cell line producing themonoclonal antibody was deposited with the Korean Cell Line ResearchFoundation, an international depository authority located atYungun-Dong, Jongno-Gu, Seoul, Korea, on Oct. 9, 2009, under AccessionNo. KCLRF-BP-00220 according to the Budapest Treaty (see Korean PatentLaid-Open Publication No. 2011-0047698).

1.1.4. Production and Purification of a Monoclonal Antibody

The hybridoma cell line obtained in Reference Example 1.1.3 was culturedin a serum-free medium, and the monoclonal antibody (AbF46) was producedand purified from the cell culture.

First, the hybridoma cells cultured in 50 mL of a medium (DMEM)supplemented with 10% (v/v) fetal bovine serum (FBS) were centrifugedand the cell pellet was washed twice or more with 20 mL of PBS to removethe FBS therefrom. Then, the cells were resuspended in 50 mL of DMEM andincubated for 3 days at 37° C. in a CO₂ incubator.

After the cells were removed by centrifugation, the supernatant wasstored at 4° C. before use or immediately used for the separation andpurification of the antibody. An AKTA system (GE Healthcare) equippedwith an affinity column (Protein G agarose column; Pharmacia, USA) wasused to purify the antibody from 50 to 300 mL of the supernatant,followed by concentration with an filter (Amicon). The antibody wasstored in PBS before use in the following examples.

1.2. Construction of chAbF46, a Chimeric Antibody to c-Met

A mouse antibody is apt to elicit immunogenicity in humans. To solvethis problem, chAbF46, a chimeric antibody, was constructed from themouse antibody AbF46 produced in Reference Example 1.1.4 by replacingthe constant region, but not the variable region responsible forantibody specificity, with an amino sequence of the human IgG1 antibody.

In this regard, a gene was designed to include the nucleotide sequenceof “EcoRI-signal sequence-VH-NheI-CH-TGA-XhoI” (SEQ ID NO: 38) for aheavy chain and the nucleotide sequence of “EcoRI-signalsequence-VL-BsiWI-CL-TGA-XhoI” (SEQ ID NO: 39) for a light chain andsynthesized. Then, a DNA fragment having the heavy chain nucleotidesequence (SEQ ID NO: 38) and a DNA fragment having the light chainnucleotide sequence (SEQ ID NO: 39) were digested with EcoRI (NEB,R0101S) and XhoI (NEB, R0146S) before cloning into a vector from thepOptiVEC™-TOPO TA Cloning Kit enclosed in an OptiCHO™ Antibody ExpressKit (Cat no. 12762-019, Invitrogen), and a vector from the pcDNA™3.3-TOPO TA Cloning Kit (Cat no. 8300-01), respectively.

Each of the constructed vectors was amplified using Qiagen Maxiprep kit(Cat no. 12662), and a transient expression was performed usingFreestyle™ MAX 293 Expression System (Invitrogen). 293 F cells were usedfor the expression and cultured in FreeStyle™ 293 Expression Medium in asuspension culture manner. At one day before the transient expression,the cells were provided in the concentration of 5×10⁵ cells/ml, andafter 24 hours, when the cell number reached to 1×10⁶ cells/ml, thetransient expression was performed. A transfection was performed by aliposomal reagent method using Freestyle ™ MAX reagent (Invitrogen),wherein in a 15 ml tube, the DNA was provided in the mixture ratio of1:1 (heavy chain DNA:light chain DNA) and mixed with 2 ml of OptiPro™SFM (Invitrogen) (A), and in another 15 ml tube, 100 ul (microliter) ofFreestyle ™ MAX reagent and 2 ml of OptiPro™ SFM were mixed (B),followed by mixing (A) and (B) and incubating for 15 minutes. Theobtained mixture was slowly mixed with the cells provided one day beforethe transient expression. After completing the transfection, the cellswere incubated in 130 rpm incubator for 5 days under the conditions of37° C., 80% humidity, and 8% CO₂.

Afterwards, the cells were incubated in DMEM supplemented with 10% (v/v)FBS for 5 hours at 37° C. under a 5% CO₂ condition and then in FBS-freeDMEM for 48 hours at 37° C. under a 5% CO₂ condition.

After centrifugation, the supernatant was applied to AKTA prime (GEHealthcare) to purify the antibody. In this regard, 100 mL of thesupernatant was loaded at a flow rate of 5 mL/min to AKTA Prime equippedwith a Protein A column (GE Healthcare, 17-0405-03), followed by elutionwith an IgG elution buffer (Thermo Scientific, 21004). The buffer wasexchanged with PBS to purify a chimeric antibody AbF46 (hereinafterreferred to as “chAbF46”).

1.3. Construction of Humanized Antibody huAbF46 from Chimeric AntibodychAbF46

1.3.1. Heavy Chain Humanization

To design two domains H1-heavy and H3-heavy, human germline genes whichshare the highest identity/homology with the VH gene of the mouseantibody AbF46 purified in Reference Example 1.2 were analyzed. An IgBLAST (www.ncbi.nlm.nih.gov/igblast/) result revealed that VH3-71 has anidentity/identity/homology of 83% at the amino acid level. CDR-H1,CDR-H2, and CDR-H3 of the mouse antibody AbF46 were defined according toKabat numbering. A design was made to introduce the CDR of the mouseantibody AbF46 into the framework of VH3-71. Hereupon, back mutations tothe amino acid sequence of the mouse AbF46 were conducted at positions30 (S→T), 48 (V→L), 73 (D→N), and 78 (T→L). Then, H1 was further mutatedat positions 83 (R→K) and 84 (A→T) to finally establish H1-heavy (SEQ IDNO: 40) and H3-heavy (SEQ ID NO: 41).

For use in designing H4-heavy, human antibody frameworks were analyzedby a BLAST search. The result revealed that the VH3 subtype, known to bemost stable, is very similar in framework and sequence to the mouseantibody AbF46. CDR-H1, CDR-H2, and CDR-H3 of the mouse antibody AbF46were defined according to Kabat numbering and introduced into the VH3subtype to construct H4-heavy (SEQ ID NO: 42).

1.3.2. Light Chain Humanization

To design two domains H1-light (SEQ ID NO: 43) and H2-light (SEQ ID NO:44), human germline genes which share the highest identity/homology withthe VH gene of the mouse antibody AbF46 were analyzed. An Ig BLASTsearch result revealed that VK4-1 has a identity/homology of 75% at theamino acid level. CDR-L1, CDR-L2, and CDR-L3 of the mouse antibody AbF46were defined according to Kabat numbering. A design was made tointroduce the CDR of the mouse antibody AbF46 into the framework ofVK4-1. Hereupon, back mutations to the amino acid sequence of the mouseAbF46 were conducted at positions 36 (Y→H), 46 (L→M), and 49 (Y→I). Onlyone back mutation was conducted at position 49 (Y→I) on H2-light.

To design H3-light (SEQ ID NO: 45), human germline genes which share thehighest identity/homology with the VL gene of the mouse antibody AbF46were analyzed by a BLAST search. As a result, VK2-40 was selected. VLand VK2-40 of the mouse antibody AbF46 were found to have aidentity/homology of 61% at an amino acid level. CDR-L1, CDR-L2, andCDR-L3 of the mouse antibody were defined according to Kabat numberingand introduced into the framework of VK4-1. Back mutations wereconducted at positions 36 (Y→H), 46 (L→M), and 49 (Y→I) on H3-light.

For use in designing H4-light (SEQ ID NO: 46), human antibody frameworkswere analyzed. A Blast search revealed that the Vk1 subtype, known to bethe most stable, is very similar in framework and sequence to the mouseantibody AbF46. CDR-L1, CDR-L2, and CDR-L3 of the mouse antibody AbF46were defined according to Kabat numbering and introduced into the Vk1subtype. Hereupon, back mutations were conducted at positions 36 (Y→H),46 (L→M), and 49 (Y→I) on H4-light.

Thereafter, DNA fragments having the heavy chain nucleotide sequences(H1-heavy: SEQ ID NO: 47, H3-heavy: SEQ ID NO: 48, H4-heavy: SEQ ID NO:49) and DNA fragments having the light chain nucleotide sequences(H1-light: SEQ ID NO: 50, H2-light: SEQ ID NO: 51, H3-light: SEQ ID NO:52, H4-light: SEQ ID NO: 53) were digested with EcoRI (NEB, R0101S) andXhoI (NEB, R0146S) before cloning into a vector from the pOptiVEC™-TOPOTA Cloning Kit enclosed in an OptiCHO™ Antibody Express Kit (Cat no.12762-019, Invitrogen) and a vector from the pcDNA™ 3.3-TOPO TA CloningKit (Cat no. 8300-01), respectively, so as to construct recombinantvectors for expressing a humanized antibody.

Each of the constructed vectors was amplified using Qiagen Maxiprep kit(Cat no. 12662), and a transient expression was performed usingFreestyle™ MAX 293 Expression System (Invitrogen). 293 F cells were usedfor the expression and cultured in FreeStyle™ 293 Expression Medium in asuspension culture manner. At one day before the transient expression,the cells were provided in the concentration of 5×10⁵ cells/ml. After 24hours, when the cell number reached 1×10⁶ cells/ml, the transientexpression was performed. A transfection was performed by a liposomalreagent method using Freestyle™ MAX reagent (Invitrogen), wherein in a15 ml tube, the DNA was provided in the mixture ratio of 1:1 (heavychain DNA:light chain DNA) and mixed with 2 ml of OptiPro™ SFM(Invitrogen) (A), and in another 15 ml tube, 100 ul (microliter) ofFreestyle™ MAX reagent and 2 ml of OptiPro™ SFM were mixed (B), followedby mixing (A) and (B) and incubating for 15 minutes. The obtainedmixture was slowly mixed with the cells provided one day before thetransient expression. After completing the transfection, the cells wereincubated in 130 rpm incubator for 5 days under the conditions of 37°C., 80% humidity, and 8% CO₂.

After centrifugation, the supernatant was applied to AKTA prime (GEHealthcare) to purify the antibody. In this regard, 100 mL of thesupernatant was loaded at a flow rate of 5 mL/min to AKTA Prime equippedwith a Protein A column (GE Healthcare, 17-0405-03), followed by elutionwith an IgG elution buffer (Thermo Scientific, 21004). The buffer wasexchanged with PBS to purify a humanized antibody AbF46 (hereinafterreferred to as “huAbF46”). The humanized antibody huAbF46 used in thefollowing examples included a combination of H4-heavy (SEQ ID NO: 42)and H4-light (SEQ ID NO: 46).

1.4. Construction of an scFV Library of huAbF46 Antibody

For use in constructing an scFv of the huAbF46 antibody from the heavyand light chain variable regions of the huAbF46 antibody, a gene wasdesigned to have the structure of “VH-linker-VL” for each of the heavyand the light chain variable region, with the linker having the aminoacid sequence “GLGGLGGGGSGGGGSGGSSGVGS” (SEQ ID NO: 54). Apolynucleotide sequence (SEQ ID NO: 55) encoding the designed scFv ofhuAbF46 was synthesized in Bioneer and an expression vector for thepolynucleotide had the nucleotide sequence of SEQ ID NO: 56.

After expression, the product was found to exhibit specificity to c-Met.

1.5. Construction of Library Genes for Affinity Maturation

1.5.1. Selection of Target CDRs and Synthesis of Primers

The affinity maturation of huAbF46 was achieved. First, sixcomplementary determining regions (CDRs) were defined according to Kabatnumbering. The CDRs are given in Table 4, below.

TABLE 4 CDR Amino Acid Sequence CDR-H1 DYYMS(SEQ ID NO: 1) CDR-H2FIRNKANGYTTEYSASVKG(SEQ ID NO: 2) CDR-H3 DNWFAY(SEQ ID NO: 3) CDR-L1KSSQSLLASGNQNNYLA(SEQ ID NO: 10) CDR-L2 WASTRVS(SEQ ID NO: 11) CDR-L3QQSYSAPLT(SEQ ID NO: 12)

For use in the introduction of random sequences into the CDRs of theantibody, primers were designed as follows. Conventionally, N codonswere utilized to introduce bases at the same ratio (25% A, 25% G, 25% C,25% T) into desired sites of mutation. In this experiment, theintroduction of random bases into the CDRs of huAbF46 was conducted insuch a manner that, of the three nucleotides per codon in the wild-typepolynucleotide encoding each CDR, the first and second nucleotidesconserved over 85% of the entire sequence while the other threenucleotides were introduced at the same percentage (each 5%) and thatthe same possibility was imparted to the third nucleotide (33% G, 33% C,33% T).

1.5.2. Construction of a Library of huAbF46 Antibodies and Affinity forc-Met

The construction of antibody gene libraries through the introduction ofrandom sequences was carried out using the primers synthesized in thesame manner as in Reference Example 1.5.1. Two PCR products wereobtained using a polynucleotide covering the scFV of huAbF46 as atemplate, and were subjected to overlap extension PCR to give scFvlibrary genes for huAbF46 antibodies in which only desired CDRs weremutated. Libraries targeting each of the six CDRs prepared from the scFVlibrary genes were constructed.

The affinity for c-Met of each library was compared to that of thewildtype. Most libraries were lower in affinity for c-Met, compared tothe wild-type. The affinity for c-Met was retained in some mutants.

1.6. Selection of an Antibody with Improved Affinity from Libraries

After maturation of the affinity of the constructed libraries for c-Met,the nucleotide sequence of scFv from each clone was analyzed. Thenucleotide sequences thus obtained are summarized in Table 2 and wereconverted into IgG forms. Four antibodies which were respectivelyproduced from clones L3-1, L3-2, L3-3, and L3-5 were used in thesubsequent experiments.

TABLE 5 Library Clone constructed CDR Sequence H11-4 CDR-H1 PEYYMS (SEQID NO: 22) YC151 CDR-H1 PDYYMS (SEQ ID NO: 23) YC193 CDR-H1 SDYYMS (SEQID NO: 24) YC244 CDR-H2 RNNANGNT (SEQ ID NO: 25) YC321 CDR-H2 RNKVNGYT(SEQ ID NO: 26) YC354 CDR-H3 DNWLSY (SEQ ID NO: 27) YC374 CDR-H3 DNWLTY(SEQ ID NO: 28) L1-1 CDR-L1 KSSHSLLASGNQNNYLA (SEQ ID NO: 29) L1-3CDR-L1 KSSRSLLSSGNHKNYLA (SEQ ID NO: 30) L1-4 CDR-L1 KSSKSLLASGNQNNYLA(SEQ ID NO: 31) L1-12 CDR-L1 KSSRSLLASGNQNNYLA (SEQ ID NO: 32) L1-22CDR-L1 KSSHSLLASGNQNNYLA (SEQ ID NO: 33) L2-9 CDR-L2 WASKRVS (SEQ ID NO:34) L2-12 CDR-L2 WGSTRVS (SEQ ID NO: 35) L2-16 CDR-L2 WGSTRVP (SEQ IDNO: 36) L3-1 CDR-L3 QQSYSRPYT (SEQ ID NO: 13) L3-2 CDR-L3 GQSYSRPLT (SEQID NO: 14) L3-3 CDR-L3 AQSYSHPFS (SEQ ID NO: 15) L3-5 CDR-L3 QQSYSRPFT(SEQ ID NO: 16) L3-32 CDR-L3 QQSYSKPFT (SEQ ID NO: 37)

1.7. Conversion of Selected Antibodies into IgG

Respective polynucleotides encoding heavy chains of the four selectedantibodies were designed to have the structure of “EcoRI-signalsequence-VH-NheI-CH-XhoI” (SEQ ID NO: 38). The heavy chains of huAbF46antibodies were used as they were because their amino acids were notchanged during affinity maturation. In the case of the hinge region,however, the U6-HC7 hinge (SEQ ID NO: 57) was employed instead of thehinge of human IgG1. Genes were also designed to have the structure of“EcoRI-signal sequence-VL-BsiWI-CL-XhoI” for the light chain.Polypeptides encoding light chain variable regions of the fourantibodies which were selected after the affinity maturation weresynthesized in Bioneer. Then, a DNA fragment having the heavy chainnucleotide sequence (SEQ ID NO: 38) and DNA fragments having the lightchain nucleotide sequences (DNA fragment including L3-1-derived CDR-L3:SEQ ID NO: 58, DNA fragment including L3-2-derived CDR-L3: SEQ ID NO:59, DNA fragment including L3-3-derived CDR-L3: SEQ ID NO: 60, and DNAfragment including L3-5-derived CDR-L3: SEQ ID NO: 61) were digestedwith EcoRI (NEB, R0101S) and XhoI (NEB, R0146S) before cloning into avector from the pOptiVEC™-TOPO TA Cloning Kit enclosed in an OptiCHO™Antibody Express Kit (Cat no. 12762-019, Invitrogen) and a vector fromthe pcDNA™ 3.3-TOPO TA Cloning Kit (Cat no. 8300-01), respectively, soas to construct recombinant vectors for expressing affinity-maturedantibodies.

Each of the constructed vectors was amplified using Qiagen Maxiprep kit(Cat no. 12662), and a transient expression was performed usingFreestyle™ MAX 293 Expression System (Invitrogen). 293 F cells were usedfor the expression and cultured in FreeStyle™ 293 Expression Medium in asuspension culture manner. At one day before the transient expression,the cells were provided in the concentration of 5×10⁵ cells/ml, andafter 24 hours, when the cell number reached to 1×10⁶ cells/ml, thetransient expression was performed. A transfection was performed by aliposomal reagent method using Freestyle ™ MAX reagent (Invitrogen),wherein in a 15 ml tube, the DNA was provided in the mixture ratio of1:1 (heavy chain DNA:light chain DNA) and mixed with 2 ml of OptiPro™SFM (Invitrogen) (A), and in another 15 ml tube, 100 ul (microliter) ofFreestyle™ MAX reagent and 2 ml of OptiPro™ SFM were mixed (B), followedby mixing (A) and (B) and incubating for 15 minutes. The obtainedmixture was slowly mixed with the cells provided one day before thetransient expression. After completing the transfection, the cells wereincubated in 130 rpm incubator for 5 days under the conditions of 37°C., 80% humidity, and 8% CO₂.

After centrifugation, the supernatant was applied to AKTA prime (GEHealthcare) to purify the antibody. In this regard, 100 mL of thesupernatant was loaded at a flow rate of 5 mL/min to AKTA Prime equippedwith a Protein A column (GE Healthcare, 17-0405-03), followed by elutionwith an IgG elution buffer (Thermo Scientific, 21004). The buffer wasexchanged with PBS to purify four affinity-matured antibodies(hereinafter referred to as “huAbF46-H4-A1 (L3-1 origin), huAbF46-H4-A2(L3-2 origin), huAbF46-H4-A3 (L3-3 origin), and huAbF46-H4-A5 (L3-5origin),” respectively).

1.8. Construction of Constant Region- and/or Hinge Region-SubstitutedhuAbF46-H4-A1

Among the four antibodies selected in Reference Example 1.7,huAbF46-H4-A1 was found to be the highest in affinity for c-Met and thelowest in Akt phosphorylation and c-Met degradation degree. In theantibody, the hinge region, or the constant region and the hinge region,were substituted.

The antibody huAbF46-H4-A1 (U6-HC7) was composed of (a) a heavy chainincluding the heavy chain variable region of huAbF46-H4-A1, U6-HC7hinge, and the constant region of human IgG1 constant region, and (b) alight chain including the light chain variable region of huAbF46-H4-A1and human kappa constant region. The antibody huAbF46-H4-A1 (IgG2 hinge)was composed of (a) a heavy chain including a heavy chain variableregion, a human IgG2 hinge region, and a human IgG1 constant region, and(b) a light chain including the light chain variable region ofhuAbF46-H4-A1 and a human kappa constant region. The antibodyhuAbF46-H4-A1 (IgG2 Fc) was composed of (a) the heavy chain variableregion of huAbF46-H4-A1, a human IgG2 hinge region, and a human IgG2constant region, and (b) a light chain including the light variableregion of huAbF46-H4-A1 and a human kappa constant region. Hereupon, thehistidine residue at position 36 on the human kappa constant region ofthe light chain was changed to tyrosine in all of the three antibodiesto increase antibody production.

For use in constructing the three antibodies, a polynucleotide (SEQ IDNO: 63) encoding a polypeptide (SEQ ID NO: 62) composed of the heavychain variable region of huAbF46-H4-A1, a U6-HC7 hinge region, and ahuman IgG1 constant region, a polynucleotide (SEQ ID NO: 65) encoding apolypeptide (SEQ ID NO: 64) composed of the heavy chain variable regionof huAbF46-H4-A1, a human IgG2 hinge region, and a human IgG1 region, apolynucleotide (SEQ ID NO: 67) encoding a polypeptide (SEQ ID NO: 66)composed of the heavy chain variable region of huAbF46-H4-A1, a humanIgG2 region, and a human IgG2 constant region, and a polynucleotide (SEQID NO: 69) encoding a polypeptide (SEQ ID NO: 68) composed of the lightchain variable region of huAbF46-H4-A1, with a tyrosine residue insteadof histidine at position 36, and a human kappa constant region weresynthesized in Bioneer. Then, the DNA fragments having heavy chainnucleotide sequences were inserted into a vector from the pOptiVEC™-TOPOTA Cloning Kit enclosed in an OptiCHO™ Antibody Express Kit (Cat no.12762-019, Invitrogen) while DNA fragments having light chain nucleotidesequences were inserted into a vector from the pcDNA™ 3.3-TOPO TACloning Kit (Cat no. 8300-01) so as to construct vectors for expressingthe antibodies.

Each of the constructed vectors was amplified using Qiagen Maxiprep kit(Cat no. 12662), and a transient expression was performed usingFreestyle ™ MAX 293 Expression System (Invitrogen). 293 F cells wereused for the expression and cultured in FreeStyle™ 293 Expression Mediumin a suspension culture manner. At one day before the transientexpression, the cells were provided in the concentration of 5×10⁵cells/ml, and after 24 hours, when the cell number reached to 1×10⁶cells/ml, the transient expression was performed. A transfection wasperformed by a liposomal reagent method using Freestyle™ MAX reagent(Invitrogen), wherein in a 15 ml tube, the DNA was provided in themixture ratio of 1:1 (heavy chain DNA:light chain DNA) and mixed with 2ml of OptiPro™ SFM (Invitrogen) (A), and in another 15 ml tube, 100 ul(microliter) of Freestyle™ MAX reagent and 2 ml of OptiPro™ SFM weremixed (B), followed by mixing (A) and (B) and incubating for 15 minutes.The obtained mixture was slowly mixed with the cells provided one daybefore the transient expression. After completing the transfection, thecells were incubated in 130 rpm incubator for 5 days under theconditions of 37° C., 80% humidity, and 8% CO₂.

After centrifugation, the supernatant was applied to AKTA prime (GEHealthcare) to purify the antibody. In this regard, 100 mL of thesupernatant was loaded at a flow rate of 5 mL/min to AKTA Prime equippedwith a Protein A column (GE Healthcare, 17-0405-03), followed by elutionwith IgG elution buffer (Thermo Scientific, 21004). The buffer wasexchanged with PBS to finally purify three antibodies (huAbF46-H4-A1(U6-HC7), huAbF46-H4-A1 (IgG2 hinge), and huAbF46-H4-A1 (IgG2 Fc)).Among the three antibodies, huAbF46-H4-A1 (IgG2 Fc) was representativelyselected for the following examples, and referred as L3-1Y-IgG2.

Reference Example 2 Preparation of Anti-Ang2 Antibody

2.1. Ang2-Binding scFv Clone

With regard to human Ang2 polypeptide (R&D systems; Human Ang2;Accession #O15123 (hAng2); SEQ ID NO: 172), a complete human anti-Ang2antibody was prepared using a phage display scFv library (obtained fromEwha Women's University-Industry Collaboration Foundation). A detailedprotocol thereof is as follows:

The Ang2 polypeptide was applied to a Maxisorp immunotube in amounts ofabout 10 μg/ml, 1 μg/ml and 0.1 μg/ml, respectively to enrich antibodiesresponding to Ang2 through 1^(st), 2^(nd), and 3^(rd) pannings. Afterthe surface of the immunotube was blocked with about 3% (v/v) milkdissolved in PBS, about 1 ×10¹² of phage particles derived from the samephage display scFv library as described above were added to about 0.5 mlof 3% (v/v) milk, which was isothermally treated together at 37° C. for1 hours for blocking. After that, the phages blocked with milk were putinto the immunotube applied with Ang2, followed by isothermal treatmentat a room temperature for 1 hour to allow Ang2 and the phages to bebound.

After the isothermal treatment of the phages, the surface of the phageswere washed 3 to 5 times with PBS and about 0.1% (v/v) Tween 20 andthen, the bound phages were eluted using 100 mM triethanolamine. Theeluted phages were transfected into E. coli ER2537 cells (New EnglandBiolabs, USA), amplified, and then obtained to be ready for use in thenext screening step. The procedure was repeated three times by applyingthe Ang2 polypeptide to a Maxisorp immunotube in amounts of about 10μg/ml, 1 μg/ml, and 0.1 μg/ml, respectively and then, about 600 specificAng2 bound scFv clones recognizing human Ang2 (Accession #O15123) ormouse Ang2 (Accession #NP_031452) were identified when measured usingELISA (Enzyme-Linked ImmunoSorbent Assay) affinity assay (see Example2.2) as described below.

2.2. Selection of an Anti-Ang2 Antibody Producing Clone and Purificationof an Antibody

Based on binding potential with Ang2 using an ELISA format, 70 cloneswhich produce anti-Ang2 antibodies were selected from the about 600 Ang2bound scFv clones obtained in Example 2.1 above. Specifically, cloneswith high ELISA OD were selected among the clones which can bind to Ang2and inhibit Tie2 binding. Then, each clone was cultured in SB media towhich ampicillin was added up to the level of OD 600=1.0, 1 mM IPTG(Isopropyl-β-D-Thiogalactopyranoside) was injected thereto and then,periplasm fractions were collected to partially purify anti-Ang2monoclonal antibodies using NI-NTA column (QIAGEN).

2.3. Ang2:Tie2 Neutralization ELISA (Competitive ELISA)

To verify molecular interaction of the bound assemblies, a competitiveELISA was performed. A 96-well MaxiSorp™ flat-bottom plate (Nunc) wascoated with 4 μg/μl of hTie2-Fc (R&D Systems), which is a protein withthe Fc of human IgG1 bound thereto. After that, the plate was washedfive times with 0.05% (v/v) Tween-20-containing PBS (Phosphate BufferSaline) and then blocked with 1% (v/v) BSA (Bovine serum albumin;Sigma)-containing PBS at a room temperature for 2 hours.

To perform Ang2:Tie2 neutralization ELISA, the anti-Ang2 antibodies intheir scfv forms purified in Example 2.2 (1, 10, 100, and 1000 nM) wereadded to each well of the plate coated with hTie2-Fc, along with 1%(v/v) BSA and 400 ng/ml of FLAG-Tagged hAng2 and then, the plate wasallowed to react at a room temperature for 2 hours. Thereafter, theplate was washed five times with 0.05% Tween-20-containing PBS and then,an HRP-conjugated anti-FLAG antibody (SIGMA) diluted in 1% (v/v)BSA-containing PBS at 1:5,000 ratio (v/v) was added in the amount of 100μl (microliter) to each well to react at a room temperature for 1 hour,followed by washing five times with 0.1% (v/v) Tween-20-containing PBS.Finally, 100 μl (microliter) of TMB substrate (cell signal) was added toeach well of the plate to induce color development at a room temperaturefor 3 min. Then, the reaction was ceased by the addition of 50 μl of 5NH₂SO₄ solution and OD450 values were measured on a plate reader(Molecular Devices). Through them, 50% inhibition concentrations (IC50)against Ang2:Tie2 binding were obtained and shown in the following Table6.

TABLE 6 50% inhibition concentration against antibody Ang2:Tie2 binding(IC₅₀, nM) SAIT-ANG-2-AB-2-E6 18.9 SAIT-ANG-2-AB-4-H10 24.3SAIT-ANG-2-AB-8-A5 36.3 SAIT-ANG-2-AB-7-C9 39.7 SAIT-ANG-2-AB-3-D3 9.9SAIT-ANG-2-AB-4-C11 6.5 SAIT-ANG-2-AB-4-F5 10 SAIT-ANG-2-AB-4-F11 6.6

As in Table 6 above, it was confirmed that the anti-Ang2 antibodies canneutralize the binding between Ang2 and Tie2 receptors.

2.4. Binding ELISA of hAng2 and mAng2

To measure binding of the antibodies prepared above with each antigen,ELISA was performed. A 96-well MaxiSorp™ flat-bottom plate (Nunc) wascoated with 5˜20 μg/ml of human Ang2 and mouse Ang2 (Accession#NP_031452) (both, R&D Systems). After that, the plate was washed fivetimes with 0.05% (v/v) Tween-20-containing PBS and then blocked with 1%(v/v) BSA-containing PBS at a room temperature for 2 hours. Theanti-Ang2 antibodies in their scFv forms prepared above were added toeach well of the plate which was then allowed to react at a roomtemperature for 2 hours.

Thereafter, the plate was washed five times with 0.05%Tween-20-containing PBS and then, an HRP-conjugated anti-HA (HA-probeAntibody (F-7) HRP conjugated) antibody (Santacruz) diluted in 1% (v/v)BSA-containing PBS at 1:1,000 ratio (v/v) was added in the amount of 50μl to each well to react at a room temperature for 1 hour, followed bywashing five times with 0.1% (v/v) Tween-20-containing PBS. Finally, 100μl of TMB substrate (cell signal) was added to each well of the plate toinduce color development at a room temperature for 3 min and then, thereaction was ceased by the addition of 50 μl of 5N H₂SO₄ solution andOD450 values were measured on a plate reader (Molecular Devices). Byobtaining 50% binding concentrations (Kd) to human Ang2 and mouse Ang2proteins through them, the binding degrees of the anti-Ang2 antibodiesto each antigen were measured. The obtained results are shown in thefollowing Table 7.

TABLE 7 Antibody human Ang2 (Kd, nM) mouse Ang2 (Kd, nM)SAIT-ANG2-AB-2-E6 9.3 5.1 SAIT-ANG2-AB-4-H10 5.3 15.8 SAIT-ANG2-AB-8-A55.3 11.7 SAIT-ANG2-AB-7-C9 3.8 8.7 SAIT-ANG2-AB-3-D3 4.3 37.8SAIT-ANG2-AB-4-C11 4.9 28.6 SAIT-ANG2-AB-4-F5 12.1 23.8SAIT-ANG2-AB-4-F11 3 20.7

2.5. Ang2 Epitope Mapping

To identify each epitope for the anti-Ang2 antibodies obtained above,ELISA was performed using recombinant proteins in which the receptorbinding sites of Ang2 protein were artificially mutated.

A 96-well MaxiSorp™ flat-bottom plate (Nunc) was coated with 50 μl ofthe anti-Ang2 scFv selected above. After that, the plate was washed fivetimes with 0.05% (v/v) Tween-20-containing PBS and then blocked with 1%(v/v) BSA-containing PBS at a room temperature for 2 hours. 5417, Q418,P419, N421, I434, D448, A449, P452, Y460, N467, K468, or F469 residue ofAng2 was mutated with alanine and tagged with FLAG (N-DYKDDDDK-C; 1012Da) and then 250 ng of them was each added to each well to the plate,which was allowed to react at a room temperature for 2 hours.Thereafter, the plate was washed five times with 0.05%Tween-20-containing PBS and then, an HRP-conjugated anti-FLAG antibody(SIGMA) diluted in 1% (v/v) BSA-containing PBS at 1:5,000 ratio (v/v)was added in the amount of 50 μl to each well to react at a roomtemperature for 1 hour, followed by washing five times with 0.1% (v/v)Tween-20-containing PBS.

Finally, 100 μl of TMB substrate (cell signal) was added to each well ofthe plate to induce color development and then, the reaction was ceasedby the addition of 50 μl of 5N H₂SO₄ solution and OD450 values weremeasured on a plate reader (Molecular Devices). By comparing bindingwith mutated Ang2 to binding with non-mutated Ang2, each epitope for theAng2 antibodies was identified. The obtained results are shown in thefollowing Tables 8 and 9.

TABLE 8 Relative binding (%) with Mutant Ang2 compared to the bindingwith native Ang2 I434 A449 P452 Y460 N467 K468 F469 AB-2-E6 5.3 3.3 24.040.0 72.1 94.0 112.3 AB-4-H10 32.9 71.0 269.8 245.8 29.8 214.5 276.7AB-8-A5 4.0 30.5 86.5 91.6 90.7 101.0 101.5 AB-7-C9 8.7 14.5 85.0 78.186.6 97.3 89.4 AB-3-D3 93.4 100.6 97.3 95.1 90.9 95.9 96.4 AB-4-C11 7.24.8 70.0 74.3 81.7 94.8 102.3 AB-4-F5 68.6 14.0 15.0 21.8 11.3 87.4259.0 AB-4-F11 69.0 16.7 91.6 96.1 92.0 101.5 104.9 Control Antibody 193.3 95.8 95.4 86.7 94.4 86.9 3.2 (Of the above table, control antibody1 is Regeneron Ang2 antibody.)

TABLE 9 Clone # Epitope (binding region) 1 AB-2-E6, AB-7-C9, AB-4-C11I434, A449 2 AB-8-A5 I434 3 AB-4-F11 A449 4 AB-4-F5 A449, P452, N467

2.6. Human Anti-Ang2 Antibody Gene Cloning

The gene sequences of heavy chain and light chain variable regions ofmonoclonal antibodies to be produced from each clone were amplifiedusing a thermocycler (GeneAmp PCR System 9700, Applied Biosystem) fromeach antibody producing E. coli glycerol stock obtained from the aboveantibody selection results.

PCR Conditions

5 min. at 94° C.;

[1 min. at 94° C., 1 min. at 55° C., and 2 min. at 72° C.]×30 cycles;

6 min. at 72° C.;

Cooling to 4° C.

Primers: pC3X-f: 3′-GCACGACAGGTTTCCCGAC-5′(SEQ ID NO: 190), pC3X-b:3′-AACCATCGATAGCAGCACCG-5′(SEQ ID NO: 191).

The PCR products obtained from each reaction were washed with QIAquickMultiwell PCR Purification kit (Qiagen) according to the Manufacturer'sprotocol.

The PCR results obtained above were cloned and subjected to DNAsequencing by a well-known method. As a result, CDR sequences shown inthe following Table 10 and Table 11 were able to be obtained.

TABLE 10 Amino acid sequence of heavy chain CDR antibody CDRH1-KABATCDRH2-KABAT CDRH3-KABAT SAIT-ANG-2-AB-2-E6 DYAMS (SEQ ID AIYPDSGNKYYADSVARHSSDPKVKSGYYDDG NO: 109) KG (SEQ ID NO: 115) MDV (SEQ ID NO: 122)SAIT-ANG-2-AB-8-A5 DYYMS (SEQ ID GIYPSGGSTYYADSV ARDPSTLTYAGFDY(SEQ NO:110) KG (SEQ ID NO: 116) ID NO: 123) SAIT-ANG-2-AB-7-C9 NYAMS (SEQ IDAISSGGGNIYYADSVK AKSGIQPSPPSMSSAYAM NO: 111) G (SEQ ID NO: 117) DV (SEQID NO: 124) SAIT-ANG-2-AB-4-C11 DYAMS (SEQ ID SIYPDDGNTYYADSVARHTSHHTSIDGYYYYG NO: 109) KG (SEQ ID NO: 118) MDG (SEQ ID NO: 125)SAIT-ANG-2-AB-4-F5 DYDMS (SEQ ID SISHGDSNKYYADSV AKSSGIQESPPTYYYYGM NO:112) KG (SEQ ID NO: 119) DV (SEQ ID NO: 126) SAIT-ANG-2-AB-4-F11 DYAMS(SEQ ID SIYPDDGNTYYADSV AKHPVRLNLHPMYYYY NO: 109) KG (SEQ ID NO: 118)GMDV (SEQ ID NO: 127) SAIT-ANG-2-AB-4-H10 SYDMS (SEQ ID LISPDSSSIYYADSVKAKDLISFWRGGFDY(SEQ NO: 113) G (SEQ ID NO: 120) ID NO: 128)SAIT-ANG-2-AB-3-D3 DYDMS (SEQ ID GISSDDGNTYYADSV ARPTIDKYTLRGYYSYG NO:114) KG (SEQ ID NO: 121) MDV (SEQ ID NO: 129)

TABLE 11 Amino acid sequence of light chain CDR antibody CDRL1-KABATCDRL2-KABAT CDRL3-KABAT SAIT-ANG-2-AB-2-E6 SGSSSNIGNNAVN (SEQ ADSNRPS(SEQ ID GSWDYSLSG (SEQ ID NO: 130) NO: 138) ID NO: 145)SAIT-ANG-2-AB-8-A5 SGSSSNIGNNYVT (SEQ ADSHRPS (SEQ ID ATWDYSLSG (SEQ IDNO: 131) NO: 139) ID NO: 146) SAIT-ANG-2-AB-7-C9 SGSSSNIGNNDVY (SEQANSHRPS (SEQ ID GTWDYSLSG (SEQ ID NO: 132) NO: 140) ID NO: 147)SAIT-ANG-2-AB-4-C11 TGSSSNIGNNDVS (SEQ SDSKRPS (SEQ ID GSWDYSLSG (SEQ IDNO: 133) NO: 141) ID NO: 145) SAIT-ANG-2-AB-4-F5 SGSSSNIGSNAVN (SEQADSNRPS (SEQ ID GSWDYSLSG (SEQ ID NO: 134) NO: 138) ID NO: 145)SAIT-ANG-2-AB-4-F11 TGSSSNIGNNAVS (SEQ SDSQRPS (SEQ ID ATWDYSLSA (SEQ IDNO: 135) NO: 142) ID NO: 148) SAIT-ANG-2-AB-4-H10 SGSSSNIGSNYVN (SEQSDSHRPS (SEQ ID GAWDDSLSG (SEQ ID NO: 136) NO: 143) ID NO: 149)SAIT-ANG-2-AB-3-D3 TGSSSNIGSNYVS (SEQ SDNKRPS (SEQ ID GTWDDSLNG (SEQ IDNO: 137) NO: 144) ID NO: 150)

2.7. Expression and Purification of Intact Antibody

The heavy chain and light chain variable regions obtained in Example 2.6above (see Tables 12 and 13 below) were each cloned into differentvectors. The heavy chain variable region was cloned into a vectorpOPTI-VAC (Invitrogen) having a CMV promoter (cytomegalovirus promoter)and including the constant region and Fc region of human IgG1. The lightchain variable region was cloned into a vector pFUSE2-CLIg-h12(Invivogen) having a CMV promoter (cytomegalovirus promoter) andincluding the constant region of human IgG1.

In particular, the heavy chain and the vector including it were treatedwith ecorI (neb) and NheI (neb) restriction enzymes and the light chainand the vector including it were treated with ecorI (neb) and avrII(neb) restriction enzymes and then, they were ligated with a T4 DNALigase (New England Biolab) to prepare a heavy chain vector and a lightchain vector for human antibody expression including the desirableregions.

The thus obtained heavy chain vector and light chain vector weretransfected together into 293-F cells (Invitrogen). The cells werecultured in a serum-free 293-f expression medium (Invitrogen) at 37° C.and on 5 days, the culture medium was collected. As a result ofSDS-PAGE, the culture medium obtained from the culture included humanantibodies consisting of heavy chains and light chains having variableregion sequences set forth in the following Tables 12 and 13. Theculture medium containing the expressed chimeric antibodies wascentrifuged at the speed of 1000×g for 10 min to remove the remainingcells and impurities, followed by affinity chromatography using ProteinA (GE-Healthcare) having a strong affinity to antibody Fc regions topurify antibodies through a low PH elution.

The amino acid sequences and the nucleotide sequences of the heavy chainvariable regions and the light chain variable regions of the antibodiespurified above were analyzed and shown in the following Table 12 andTable 13.

TABLE 12 Amino acid sequence of heavy chain Amino acid sequence of lightchain antibody variable region variable region SAIT-ANG-2-AB-2-E6EVQLLESGGGLVQTGGSLRLSCAA QSVLTQPPSASGTPGQRVTISCSGSSGFTFSDYAMSWVRQAPGKGLEW SSNIGNNAVNWYQQLPGTAPKLLIVSAIYPDSGNKYYADSVKGRFTIS YADSNRPSGVPDRFSGSKSGTSASRDNSKNTLYLQMNSLRAEDTAVY LAISGLRSEDEADYYCGSWDYSLS YCARHSSDPKVKSGYYDDGMDVGYVFGGGTKLTVLG (SEQ ID NO: WGQGTLVAVSS (SEQ ID NO: 156) 164)SAIT-ANG-2-AB-8-A5 EVQLLESGGGLVQPGGSLRLSCAA QSVLTQPPSASGTPGQRVTISCSGSSGFTFSDYYMSWVRQAPGKGLEW SSNIGNNYVTWYQQLPGTAPKLLIVSGIYPSGGSTYYADSVKGRFTIS YADSHRPSGVPDRFSGSKSGTSASRDNSKNTLYLQMNSLRAEDTAVY LAISGLRSEDEADYYCATWDYSLS YCARDPSTLTYAGFDYWGQGTLVGYVFGGGTKLTVLG (SEQ ID NO: TVSS (SEQ ID NO: 157) 165) SAIT-ANG-2-AB-7-C9EVQLLESGGGLVQPGGSLRLSCAA QSVLTQPPSASGTPGQRVTISCSGSSGFTFSNYAMSWVRQAPGKGLEW SSNIGNNDVYWYQQLPGTAPKLLIVSAISSGGGNIYYADSVKGRFTISR YANSHRPSGVPDRFSGSKSGTSASDNSKNTLYLQMNSLRAEDTAVYY LAISGLRSEDEADYYCGTWDYSLS CAKSGIQPSPPSMSSAYAMDVWGGYVFGGGTKLTVLG (SEQ ID NO: QGTLVTVSS (SEQ ID NO: 158) 166)SAIT-ANG-2-AB-4-C11 EVQLLESGGGLVQPGGSLRLSCAA QSVLTQPPSASGTPGQRVTISCTGSSGFTFSDYAMSWVRQAPGKGLEW SSNIGNNDVSWYQQLPGTAPKLLIVSSIYPDDGNTYYADSVKGRFTIS YSDSKRPSGVPDRFSGSKSGTSASLRDNSKNTLYLQMNSLRAEDTAVY AISGLRSEDEADYYCGSWDYSLSG YCARHTSHHTSIDGYYYYGMDGYVFGGGTKLTVLG (SEQ ID NO: WGQGTLVTVSS (SEQ ID NO: 159) 167)SAIT-ANG-2-AB-4-F5 EVQLLESGGGLVQPGGSLRLSCAA QSVLTQPPSASGTPGQRVTISCSGSSGFTFSDYDMSWVRQAPGKGLEW SSNIGSNAVNWYQQLPGTAPKLLIVSSISHGDSNKYYADSVKGRFTIS YADSNRPSGVPDRFSGSKSGTSASRDNSKNTLYLQMNSLRAEDTAVY LAISGLRSEDEADYYCGSWDYSLS YCAKSSGIQESPPTYYYYGMDVWGYVFGGGTKLTVLG (SEQ ID NO: GQGTLVTVSS (SEQ ID NO: 160) 168)SAIT-ANG-2-AB-4-F11 EVQLLESGGGLVQTGGSLRLSCAA QSVLTQPPSASGTPGQRVTISCTGSSGFTFSDYAMSWVRQAPGKGLEW SSNIGNNAVSWYQQLPGTAPKLLIVSSIYPDDGNTYYADSVKGRFTIS YSDSQRPSGVPDRFSGSKSGTSASLRDNSKNTLYLQMNSLRAEDTAVY AISGLRSEDEADYYCATWDYSLSA YCAKHPVRLNLHPMYYYYGMDYVFGGGTKLTVLG (SEQ ID NO: VWGQGTLVTVSS (SEQ ID NO: 161) 169)SAIT-ANG-2-AB-4-H10 EVQLLESGGGLVQPGGSLRLSCAA QSVLTQPPSASGTPGQRVTISCSGSSGFTFSSYDMSWVRQAPGKGLEW SSNIGSNYVNWYQQLPGTAPKLLIVSLISPDSSSIYYADSVKGRFTISR YADSNRPSGVPDRFSGSKSGTSASDNSKNTLYLQMNSLRAEDTAVYY LAISGLRSEDEADYYCGSWDYSLS CAKDLISFWRGGFDYWGQGTLVGYVFGGGTKLTVLG (SEQ ID NO: TVSS (SEQ ID NO: 162) 170) SAIT-ANG-2-AB-3-D3EVQLLESGGGLVQPGGSLRLSCAA QSVLTQPPSASGTPGQRVTISCTGSSGFTFSDYDMSWVRQAPGKGLEW SSNIGSNYVSWYQQLPGTAPKLLIVSGISSDDGNTYYADSVKGRFTIS YSDNKRPSGVPDRFSGSKSGTSASRDNSKNTLYLQMNSLRAEDTAVY LAISGLRSEDEADYYCGTWDDSL YCARPTIDKYTLRGYYSYGMDVNGYVFGGGTKLTVLG (SEQ ID NO: WGQGTLVTVSS (SEQ ID NO: 163) 171) (Of theabove Table, portions marked in bold types are CDR1, CDR2, and CDR3 inorder.)

TABLE 13 Amino acid sequence of heavy chain Amino acid sequence of lightchain variable antibody variable region region SAIT-ANG-2-GAAGTGCAGCTTCTGGAATCAGGCG CAGTCAGTCCTGACACAGCCCCCTAG AB-2-E6GTGGACTGGTGCAGCCAGGAGGCAG TGCTTCCGGAACCCCTGGGCAGAGGGCCTCAGGCTGTCTTGCGCAGCCAGC TGACCATCTCATGCTCAGGTAGCTCCGGATTTACCTTCTCCGATTACGCCAT AGCAACATTGGAAACAATGCAGTTAAGAGCTGGGTTAGACAGGCCCCTGGC TTGGTATCAGCAACTGCCCGGGACCGAAGGGGCTGGAGTGGGTCAGTGCCA CCCCAAAGCTTCTGATCTACGCTGATTTTACCCCGACTCCGGGAATAAGTA AGTAATAGACCATCTGGAGTGCCTGATTACGCTGACTCTGTGAAAGGTAGA CAGATTCAGTGGTTCGAAAAGCGGCATTCACTATCTCAAGAGACAACTCCA CTTCTGCATCCTTGGCCATTTCTGGCTAAAATACATTGTACTTACAGATGAA TAAGATCTGAAGATGAGGCCGACTATCTCACTGCGCGCTGAGGATACAGCA TACTGTGGCTCTTGGGACTACTCCCTGGTGTATTATTGTGCGCGGCACTCGA AGCGGATATGTGTTTGGGGGCGGAACGTGATCCTAAGGTCAAAAGCGGATA TAAGCTCACAGTCCTAGGC (SEQ IDCTATGACGACGGCATGGATGTTTGG NO: 174) GGCCAAGGGACTCTCGTAACCGTGT CTTCT (SEQID NO: 173) SAIT-ANG-2- GAGGTCCAGCTGCTCGAATCAGGCGCAAAGTGTTCTCACACAGCCGCCATC AB-8-A5 GTGGGCTGGTGCAGCCAGGCGGCTCCGCTTCCGGGACCCCTGGACAGAGAG CCTGAGGTTAAGTTGCGCCGCTTCTGTGACCATCAGTTGTAGTGGCTCTTCGA GCTTTACATTTAGCGATTATTACATGGCAATATTGGCAATAACTATGTGACA TCCTGGGTCCGCCAGGCCCCCGGGATGGTATCAGCAGCTTCCTGGAACAGC AAGGTCTGGAGTGGGTGAGCGGAATCCCCAAACTGCTCATCTATGCCGACA TTACCCTTCCGGGGGAAGCACCTATGCCACAGACCATCAGGTGTCCCCGAT TACGCGGATTCTGTAAAGGGTAGATAGATTTTCTGGGTCAAAGTCAGGAAC TCACTATCTCAAGAGACAATTCTAATAGCGCAAGCCTGGCCATTTCTGGAT GAATACCCTGTATTTGCAGATGAACTAAGGTCCGAGGACGAAGCTGATTAC AGTCTTAGAGCCGAAGACACAGCAGTATTGCGCAACTTGGGACTACTCTCTG TTTATTATTGTGCAAGAGACCCCAGTTCTGGTTACGTGTTCGGCGGCGGAAC ACTCTAACCTACGCTGGCTTCGATTACAAGTTGACGGTCCTAGGC (SEQ ID CTGGGGACAAGGAACGCTCGTGACA NO: 176) GTGTCAAGC(SEQ ID NO: 175) SAIT-ANG-2- GAGGTGCAACTCCTGGAGTCAGGAGCAGAGCGTCCTGACACAACCTCCATC AB-7-C9 GCGGCCTGGTCCAGCCCGGCGGGAGCGCTTCTGGGACGCCTGGACAGAGAG TCTTAGACTCTCGTGTGCCGCAAGCTGACAATTTCTTGCAGCGGCTCATCTT GGGTTTACATTCAGTAACTACGCCACAAATATTGGAAACAATGACGTTTAT TGTCCTGGGTCAGACAGGCTCCTGGTGGTACCAGCAGCTCCCAGGGACCGC AAAGGGACTGGAATGGGTTTCTGCCCCCAAAGCTGCTGATCTATGCAAACT ATTAGCTCCGGCGGAGGTAATATCTCACACAGACCCAGCGGAGTGCCCGAT ATTACGCTGATTCCGTTAAAGGGAGAGATTCAGTGGATCCAAATCCGGCAC GTTTACAATCTCTCGGGATAACAGCTAGTGCCAGCTTGGCAATCTCGGGGC AAAAATACTTTGTATCTGCAGATGATGAGATCTGAAGACGAGGCTGATTAC ATAGCTTAAGAGCCGAAGACACTGCTATTGTGGTACCTGGGATTACTCCTTA AGTGTACTACTGCGCGAAGAGCGGTAGTGGTTACGTGTTTGGCGGGGGCAC ATTCAACCCTCTCCACCATCCATGTCTAAGCTTACCGTCCTAGGC (SEQ ID ATCAGCTTATGCAATGGACGTATGG NO: 178)GGGCAGGGCACCCTGGTGACCGTGA GTTCT (SEQ ID NO: 177) SAIT-ANG-2-GAAGTACAGCTGCTGGAGTCGGGTG CAGTCAGTCCTGACTCAGCCACCCTC AB-4-C11GTGGACTGGTTCAGCCAGGAGGCTC CGCAAGCGGGACACCTGGACAAAGAATTAAGGCTGAGCTGCGCCGCAAGC GTTACTATCTCTTGCACCGGGTCAAGCGGTTTCACTTTTTCTGATTATGCTAT TCCAATATCGGTAACAATGATGTGAGGTCCTGGGTCAGACAGGCCCCTGGG TTGGTACCAGCAGTTACCAGGCACCGAAGGGACTCGAGTGGGTCTCAAGTA CCCCGAAACTGCTTATTTACTCAGACTTTACCCCGACGATGGAAATACCTA AGCAAAAGACCCTCTGGCGTGCCTGACTATGCCGATAGCGTGAAGGGGCGC CAGATTCTCAGGAAGCAAGAGTGGCATTTACAATCTCTAGAGATAATTCTAA CGTCTGCTTCCTTGGCCATTTCGGGTCAAACACCCTGTACCTTCAAATGAAC TGAGATCCGAGGACGAAGCTGATTATTCATTGCGGGCAGAAGACACAGCGG TATTGTGGAAGCTGGGATTATAGTCTTGTACTATTGTGCTAGACACACGTCC GTCTGGCTACGTGTTTGGGGGCGGAACACCATACCAGCATCGACGGCTACT CCAAGCTCACAGTCCTAGGC (SEQ IDATTATTACGGGATGGACGGCTGGGG NO: 180) CCAGGGCACTCTCGTGACAGTGTCC AGT (SEQ IDNO: 179) IT-ANG-2-AB- GAGGTGCAGTTGCTCGAGTCCGGGGCAGTCTGTGTTGACCCAGCCCCCTTCT 4-F5 GTGGCCTGGTGCAGCCAGGAGGAAGGCATCTGGCACCCCCGGACAGAGAGT CCTGAGACTGAGCTGCGCAGCCTCACACTATAAGTTGTTCTGGTAGCTCCTC GGTTTCACATTCTCCGATTACGACATAAATATCGGCTCAAACGCCGTGAATT GTCCTGGGTTAGGCAAGCCCCCGGCGGTACCAGCAATTACCAGGAACAGCT AAGGGGCTGGAATGGGTAAGCTCTACCTAAGCTGCTTATCTATGCAGACAG TCAGCCACGGCGACAGTAACAAATATAACAGACCAAGCGGCGTTCCTGATA TTATGCAGACTCTGTTAAGGGACGGGATTCTCAGGCTCCAAGTCCGGGACT TTTACCATTTCACGCGATAACTCAAAAGTGCCTCGCTGGCTATTAGCGGTCTC GAATACACTGTACCTTCAAATGAATAGAAGTGAAGATGAGGCCGATTACTA AGTCTCAGAGCTGAAGATACCGCCGTTGCGGAAGCTGGGACTACTCCCTGA TGTATTACTGTGCTAAATCGTCCGGAGCGGCTATGTGTTTGGAGGAGGGACA ATCCAGGAGAGTCCCCCTACTTATT AAACTCACCGTCCTAGGC(SEQ ID NO: ACTACTATGGGATGGATGTGTGGGG 182) CCAGGGCACCCTGGTCACTGTCTCTTCTGCTAGC (SEQ ID NO: 181) SAIT-ANG-2- GAGGTGCAACTGCTGGAGAGTGGTGCAGTCTGTGTTAACACAACCTCCAAG AB-4-F11 GGGGCCTTGTTCAGCCCGGCGGATCTGCATCCGGAACGCCGGGCCAGAGAG CTTGAGGCTGTCATGCGCTGCGTCTGTGACTATCAGCTGCACCGGCAGCTCG GCTTTACTTTCAGCGATTACGCAATGTCCAATATCGGTAACAACGCAGTTAG AGTTGGGTGAGACAGGCTCCAGGAATTGGTACCAGCAGCTTCCCGGCACAG AAGGCCTGGAATGGGTCAGCTCCATCTCCAAAGCTCTTGATTTACAGCGATT TTATCCTGACGATGGTAACACATATTCACAAAGACCTAGTGGTGTCCCCGAT ACGCCGACAGCGTAAAAGGACGGTTAGATTTTCTGGGAGTAAGAGCGGGAC CACCATCTCTCGCGATAATTCTAAGCAGTGCCTCCCTGGCTATATCAGGAC AACACCCTGTATCTCCAGATGAATATGAGATCTGAAGATGAGGCTGACTAT GCCTGAGAGCAGAAGACACCGCCGTTACTGTGCCACTTGGGACTATTCACTC GTACTACTGTGCCAAGCATCCTGTGTCTGCCTATGTGTTCGGGGGCGGAAC AGATTAAACCTGCACCCAATGTACT CAAACTGACAGTCCTAGGC(SEQ ID ATTATTACGGCATGGACGTTTGGGG NO: 184) GCAGGGGACACTCGTGACTGTCTCC TCA(SEQ ID NO: 183) SAIT-ANG-2- GAGGTTCAGTTGCTGGAGAGTGGCGCAGAGCGTGCTCACCCAACCTCCCAG AB-4-H10 GCGGCTTAGTGCAGCCAGGTGGCAGTGCATCCGGAACGCCTGGTCAGAGAG CCTGCGCCTTTCTTGTGCCGCCAGTGTGACAATTAGCTGCTCAGGGTCTTCCT GGTTTACCTTCTCCTCCTACGACATGCTAACATCGGGTCCAATTATGTCAATT AGCTGGGTGCGGCAGGCTCCCGGCAGGTATCAGCAGTTGCCAGGTACAGCT AAGGTCTTGAATGGGTGTCACTGATCCCAAACTGCTGATCTACAGTGATTC CAGCCCTGACAGTTCCTCAATCTATTCCACAGACCTAGCGGCGTTCCAGACA ATGCAGATTCAGTCAAGGGAAGATTGATTTAGCGGATCCAAGTCGGGAACT TACCATAAGCAGAGATAATTCCAAGTCTGCAAGCCTCGCTATTTCTGGCCTG AATACTCTGTACCTACAGATGAACTAGAAGTGAGGACGAAGCCGATTATTA CGCTCAGAGCCGAAGATACCGCAGTCTGTGGGGCCTGGGACGATTCATTAT CTACTACTGCGCTAAAGACCTGATTTCAGGATACGTGTTCGGAGGCGGCACC CTTTCTGGAGGGGGGGATTCGACTA AAGCTTACTGTCCTAGGC(SEQ ID NO: TTGGGGGCAAGGAACACTCGTAACA 186) GTGTCTAGC (SEQ ID NO: 185)SAIT-ANG-2- GAGGTACAGCTGCTGGAAAGTGGGG CAGTCAGTGCTGACACAGCCTCCAAG AB-3-D3GCGGTCTGGTGCAGCCAGGGGGAAG CGCTTCCGGGACACCTGGACAAAGAGCCTCCGGCTTTCATGCGCCGCAAGC TTACCATTTCGTGCACCGGATCCTCCTGGCTTTACATTCAGTGACTATGACAT CAAACATCGGTAGCAATTATGTGTCTGAGTTGGGTCCGACAAGCCCCCGGA TGGTACCAGCAGCTCCCCGGGACTGCAAGGGCCTGGAGTGGGTGTCTGGAA CCCCAAACTCTTGATCTACAGCGACATCTCCTCCGATGACGGCAATACTTAT ACAAGAGACCATCTGGTGTGCCTGATTACGCTGACTCCGTTAAAGGTAGGT AGATTCAGTGGGAGTAAGTCAGGAACTCACCATCTCTCGCGATAACTCTAA GAGTGCCTCTCTGGCTATTTCTGGCCTAAACACCCTCTACCTGCAGATGAAT GAGAAGCGAAGATGAGGCAGACTATTAGCTTGAGGGCAGAAGATACGGCTG ATTGTGGCACCTGGGATGACTCCCTGTCTACTATTGTGCCAGACCTACAATT AATGGCTACGTCTTTGGCGGCGGAACGACAAGTACACATTAAGAGGGTATT AAAACTTACTGTCCTAGGC(SEQ IDATTCATACGGCATGGATGTTTGGGG NO: 188) ACAGGGAACTCTAGTGACCGTGTCC AGC (SEQ IDNO: 187)

2.8. Ang2:Tie2 Neutralization ELISA (Competitive ELISA)

To verify molecular interaction of the bound assemblies, a competitiveELISA was performed. A 96-well MaxiSorp™ flat-bottom plate (Nunc) wascoated with 4 μg/μl of hTie2-Fc (R&D Systems), which is a protein withthe Fc of human IgG1 bound thereto. After that, the plate was washedfive times with 0.05% (v/v) Tween-20-containing PBS (Phosphate BufferSaline) and then blocked with 1% (v/v) BSA (Bovine serum albumin;Sigma)-containing PBS at a room temperature for 2 hours.

To perform Ang2:Tie2 neutralization ELISA, the anti-Ang2 antibodies intheir IgG forms purified in Example 2 (0.00, 0.01, 0.1, 1, 10, 100, and1000 nM) were added to each well of the plate coated with hTie2-Fc,along with 1% (v/v) BSA and 400 ng/ml of FLAG-Tagged hAng2 and then, theplate was allowed to react at a room temperature for 2 hours.Thereafter, the plate was washed five times with 0.05%Tween-20-containing PBS and then, an HRP-conjugated anti-FLAG antibody(SIGMA) diluted in 1% (v/v) BSA-containing PBS at 1:5,000 ratio (v/v)was added in the amount of 100 μl to each well to react at a roomtemperature for 1 hour, followed by washing five times with 0.1% (v/v)Tween-20-containing PBS. Finally, 100 μl of TMB substrate (cell signal)was added to each well of the plate to induce color development at aroom temperature for 3 min. Then, the reaction was ceased by theaddition of 50 μl of 5N H₂SO₄ solution and OD450 values were measured ona plate reader (Molecular Devices). Through them, 50% inhibitionconcentrations (IC50) against Ang2:Tie2 binding were obtained and shownin the following Table 14.

TABLE 14 50% inhibition concentration against antibody Ang2:Tie2 binding(IC₅₀, nM) SAIT-ANG-2-AB-2-E6 0.605 SAIT-ANG-2-AB-4-H10 0.417SAIT-ANG-2-AB-8-A5 0.341 SAIT-ANG-2-AB-7-C9 0.392 SAIT-ANG-2-AB-3-D30.44 SAIT-ANG-2-AB-4-C11 0.421 SAIT-ANG-2-AB-4-F5 1.525SAIT-ANG-2-AB-4-F11 0.37

As in Table 14 above, it was confirmed that the anti-Ang2 antibodies canneutralize binding between Ang2 and Tie2 receptors.

2.9. Binding ELISA of mAng2

To measure binding of the antibodies prepared above with each antigen,ELISA was performed. A 96-well MaxiSorp™ flat-bottom plate (Nunc) wascoated with 5˜20 μg/ml of human Ang2 and mouse Ang2 (Accession#NP_031452) (both, R&D Systems). After that, the plate was washed fivetimes with 0.05% (v/v) Tween-20-containing PBS and then blocked with 1%(v/v) BSA-containing PBS at a room temperature for 2 hours. Theanti-Ang2 antibodies in their IgG forms prepared above (0.001, 0.01,0.1, 1, 10, 100, and 1000 nM) were added to each well of the plate whichwas then allowed to react at a room temperature for 2 hours.

Thereafter, the plate was washed five times with 0.05%Tween-20-containing PBS and then, an HRP-conjugated anti-human FC(Anti-hFc-HRP conjugated) antibody (SIGMA) diluted in 1% (v/v)BSA-containing PBS at 1:5,000 ratio (v/v) was added in the amount of 50μl to each well to react at a room temperature for 1 hour, followed bywashing five times with 0.1% (v/v)

Tween-20-containing PBS. Finally, 100 μl of TMB substrate (cell signal)was added to each well of the plate to induce color development at aroom temperature for 3 min and then, the reaction was ceased by theaddition of 50 μl of 5N H₂SO₄ solution and OD450 values were measured ona plate reader (Molecular Devices). By obtaining 50% bindingconcentrations (Kd) to human Ang2 and mouse Ang2 proteins through them,the binding degrees of the anti-Ang2 antibodies to each antigen weremeasured. The obtained results are shown in the following Table 15.

TALE 15 antibody mouse Ang2 (Kd, nM) SAIT-ANG-2-AB-2-E6 0.35SAIT-ANG-2-AB-4-H10 0.21 SAIT-ANG-2-AB-8-A5 0.19 SAIT-ANG-2-AB-3-D3 0.30SAIT-ANG-2-AB-4-C11 0.28 SAIT-ANG-2-AB-4-F11 0.36

2.10. Antigen Affinity (Kd values) Measurement Using Surface PlasmonResonance (SPR) Method

To measure accurate affinities toward an anti-Ang2 antigen, antigenaffinities were measured by a SPR method using a BIAcore T100 (GEHealthcare). The SPR method uses refractive index change of light whichpasses a sensor chip according to the state of materials coated onto thesensor chip, and if an antigen or an antibody is flowed onto a chipcoated with the antigen or antibody, it causes changes in refractiveindex due to their binding and Kd values are thus calculated from themeasured values.

First, anti-His antibody was immobilized on a CMS sensor chip (GEhealthcare) up to 8,000 RU levels using a pH 5.0 acetate solution and anamine coupling kit (GE Healthcare). 6 μg/ml of a recombinant hAng2(C-His, R&D Systems) protein was flowed onto the chip to be captured at100 to 200 RU levels. The antibodies obtained in Example 2 above werediluted serially to twice each time starting from 100 nM concentrationand each of them was flowed onto the chip to allow them to be bound to(on), dissociated from (off), and regenerated (using 10 mM NaOHsolution) from the antigen captured on the sensor chip, thereby tomeasure antigen-antibody affinities. The results are as shown in thefollowing Table 16.

TABLE 16 Affinity antibody On rate (l/Ms) Off Rate (l/s) (Kd, nM)SAIT-ANG-2-AB-2-E6 1.220 × 10⁶ 7.950 × 10⁻⁴ 0.65 SAIT-ANG-2-AB-4-H102.812 × 10⁶ 3.328 × 10⁻⁴ 0.118 SAIT-ANG-2-AB-8-A5 4.396 × 10⁶ 3.266 ×10⁻⁴ 0.074 SAIT-ANG-2-AB-7-C9 1.785 × 10⁶ 3.661 × 10⁻⁴ 0.205SAIT-ANG-2-AB-3-D3 1.162 × 10⁶ 5.461 × 10⁻⁴ 0.47 SAIT-ANG-2-AB-4-C118.327 × 10⁵ 4.899 × 10⁻⁴ 0.588 SAIT-ANG-2-AB-4-F5 1.895 × 10⁶ 0.0011170.589 SAIT-ANG-2-AB-4-F11 1.765 × 10⁶ 7.774 × 10⁻⁴ 0.44

2.11. Test of Ang2 Receptor Inhibitory Activity of Ang2 Antibodies

As Ang2 induces a change in vascular endothelial cells by binding to aTie2 receptor expressed in the vascular endothelial cells to induce thephosphorylation of the receptor and activate it, the Ang2 inhibitoryactivities of the anti-Ang2 antibodies were verified through thefunctional analysis of the antibodies using a cell-based assay.

For this, 1×10⁶ of HUVEC (ATCC) cells (Kim et al., Biochim BioohysActa., 2009) were cultured in a 60 mm culture dish using 5% (v/v) FBS(Gibco)-added EGM-2 (Endothelial growth media) media (Lonza) at 37° C.and when they reached 80˜90% confluency, the media were replaced withserum-free EGM-2 media and cultured at 37° C. for 16 hours. The dish waswashed once with PBS and after the replacement with 0.1 nM sodiumorthovanadate-mixed EGM-2 media, they were further cultured for 10 min.After washed once with PBS, the cultured cells were treated with amixture prepared by mixing the anti-Ang2 antibodies prepared in Example2 at a 10 μg/ml concentration with 2 μg/ml human Ang2 protein (R&Dsystems) and letting them stand for 20 min and further cultured for 10min.

The cells were washed using a cold PBS, treated with 300 μl of lysisbuffer (Roche), collected to a tube to allow them to be dissolved at 4°C. for 30 min. and then, centrifuged at 13,000 rpm for 15 min. tomeasure a supernatant. 2μg of anti Tie2 antibody (R&D system) was addedto 0.5 mg of a cell lysate, which was then overnight reacted at 4° C.and then subjected to immunoprecipitation by the addition of protein Abead (GE Healthcare) thereto.

The reactant obtained above was centrifuged at 13,000 rpm for 15 min. toobtain a pellet, which was washed two to three times with lysis buffer(Roche), added to a sample buffer (Invitrogen) mixed with a reducingagent, and boiled at 95° C. for 5 min., and then, applied to NuPAGENovex 4-12% Bis-Tris gel (Invitrogen) and transferred ontoNitrocellulose membrane (Invitrogen).

To verify the presence of the phosphorylation of Tie2, the above blotswere blocked with PBST mixed with 3% (v/v) skim milk (Sigma) for 30 min.and identified using an HRP-conjugated anti-phospho tyrosine antibody(Millipore). For Tie2 identification, the blots were reacted in astripping buffer (Thermo) for 15 min and then blocked again andidentified using an anti Tie2 antibody (Santa cruz). After bandintensities were measured using Image J software(http://rsb.info.nih.gov/ij/index.html), the inhibitory degrees of Tie2phosphorylation after the treatment of the anti-Ang2 antibodies werecalculated in relative % against the Ang2 single treatment group, andthe results are shown in the following Table 17.

TABLE 17 Antibody (treated amount: 50 nM) Tie2 phosphorylationinhibition (%) SAIT-ANG-2-AB-4-H10 69% SAIT-ANG-2-AB-8-A5 60%SAIT-ANG-2-AB-2-E6 62%

2.12. Inhibition Test of Binding between Ang2 and Integrin

An ELISA plate was coated with a diluting solution of integrin(alpha5beta1 (α5β1 (α5: NCBI Accession No. P08648, (31: NCBI AccessionNo. P05556); R&D systems) protein diluted in PBS at a concentration of 5μg/ml (18 hours, 4° C.) and then blocked with 1% (v/v) BSA at a roomtemperature for 2 hours. Thereafter, the plate was treated with Ang2protein (FLAG-Ang2, 0.05 ml of Ang2 protein solution diluted in PBS at aconcentration of 10 μg/ml) tagged with a FLAG sequence (DYKDDDDK, Sigma)at N-terminal and an antibody (0.05 ml of antibody solution diluted inPBS at a concentration of 10 μg/ml) at the same time, incubated at aroom temperature for 2 hours, and washed five times with PBS-t (0.1%(v/v) triton X-100 in PBS). After that, an anti-FLAG antibody (Sigma)conjugated with horseradish peroxidase (HRP) was added to react, and theplate was washed again five times with PBS-t. Bindings between Ang2 andthe above the integrin were identified indirectly by measuring theamounts of the anti-FLAG antibody remaining in the ELISA plate via colordevelopment using TMB (3,3,5,5-tetramethylbenzidine) as a substrate ofHRP. As positive controls, control antibody 1 (Regeneron Ang2 antibody)and control antibody 2 (Astra Zeneca Ang2 antibody) were used. Also, toshow the background, a group coated with BSA instead of integrin wasused.

The results obtained above were shown in FIG. 2. As seen in FIG. 2, theanti-Ang2 antibodies provided in the invention remarkably suppressedbinding between integrin and Ang2.

Example 1 Preparation of an Anti-c-Met/anti-Ang2 Bispecific Antibody

The anti-Ang2 scFv obtained on Reference Example 2 was fused to theC-terminus of Fc of the anti-c-Met antibody L3-1Y-IgG2 prepared inReference Example 1, to prepare a bispecific antibody.

The heavy chain part of L3-1Y-IgG2 antibody which was used in thebispecific antibody cloning was prepared as follows. A DNA fragmentencoding the heavy chain of the anti-c-Met antibody was synthesized bydeleting the part encoding C-terminus part from position 1393 of SEQ IDNO: 67 and inserting “ggcggtggtggttccggaggcggcggatcc” instead of thedeleted part (Bioneer Corporation). Thereafter, the DNA fragment wasligated to a vector from the pOptiVEC™-TOPO TA Cloning Kit included inOptiCHO™ Antibody Express Kit (Cat no. 12762-019) (Invitrogen).

An anti-Ang2 scFv was prepared by linking the heavy chain variableregion (SEQ ID NO: 162) and the light chain variable region (SEQ ID NO:170) of anti-Ang2 antibody 4-H10 via a peptide linker (GGGGS)₂. Inparticular, a nucleotide sequence (SEQ ID NO: 185) encoding the heavychain variable region of anti-Ang2 antibody 4-H10, a nucleotide sequence(SEQ ID NO: 186) encoding the light chain variable region of anti-Ang2antibody 4-H10, and a nucleotide sequence encoding the peptide linkerwere all synthesized by Bioneer Corporation (in the form including aN-terminal BamHI restriction site and C-terminal XhoI restriction site).

Then, the obtained anti-Ang2 scFv was cloned in the prepared L3-1Y-IgG2containing vector using restriction enzymes BamHI and XhoI, to constructan expression vector for the heavy chain of the bispecific antibody.

Each region of the amino acid sequence (SEQ ID NO: 189) encoding theheavy chain of the bispecific antibody (wherein the heavy chain of thec-Met antibody and the anti-Ang2 scFv are linked through the peptidelinker) is summarized in following Table 18:

TABLE 18 SEQ ID NO: 189 N-terminus → C-terminus Signal peptideMEWSWVFLVTLLNGIQC Heavy chain variable region of anti-c-MetEVQLVESGGGLVQPGGSLRLSCAASGFTFTDYYMSWVRQAP antibodyGKGLEWLGFIRNKANGYTTEYSASVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDNWFAYWGQGTLVTVSS Fc (IgG2)ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPGPeptide linker (linking the C-terminus of GGGGSGGGGS Fcand the anti-Ang2scFv) Heavy chain variable region of the anti-EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRLAPG Ang2 scFvKGLEWVSLISPDSSSIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDLISFWRGGFDYWGQGTLVTVSS Peptide linker (linking the heavychain GGGGSGGGGS variable region and the light chain variable region ofthe anti-Ang2 scFv) Light chain variable region of the anti-QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNYVNWYQQLPGT Ang2 scFvAPKLLIYSDSHRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCGAWDDSLSGYVFGGGTKLTVLGQAGQHHHHHHGAYPYD VPDYAS (CDRs are underlined)

In addition, the DNA fragment encoding the light chain of the anti-c-Metantibody was synthesized so that it has the nucleotide sequence of SEQID NO: 69 (Bioneer Corporation). The DNA fragment encoding the lightchain of the anti-c-Met antibody was inserted into a vector from thepcDNA™ 3.3-TOPO TA Cloning Kit (Cat no. 8300-01) included in OptiCHO™Antibody Express Kit (Cat no. 12762-019) (Invitrogen), to construct anexpression vector for the light chain of the anti-c-Met antibody.

Each region of the amino acid sequence (SEQ ID NO: 68) encoding thelight chain of the anti-c-Met antibody is summarized in following Table19:

TABLE 19 SEQ ID NO: 68 N-terminus → C-terminus Signal peptideMDSQAQVLMLLLLSVSGTCG Light chain DIQMTQSPSSLSASVGDRVTITCKSSQSLLAvariable region SGNQNNYLAWYQQKPGKAPKMLIIWASTR of theVSGVPSRFSGSGSGTDFTLTISSLQPEDFATYY anti-c-MetCQQSYSRPYTFGQGTKVEIKRTVAAPSVFIFPPS antibodyDEQLKSGTASVVCLLNNFYPREAKVQWKVDNA LQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF NRGEC (CDRs are underlined)

Each of the constructed expression vectors for the heavy chain and thelight chain was amplified using Qiagen Maxiprep kit (Cat no. 12662), anda temporary expression was performed using Freestyle™ MAX 293 ExpressionSystem (Invitrogen). For the expression, 293F cells were used andsubjected to a suspension culture using FreeStyle™ 293 ExpressionMedium. One day before the temporary expression, the cells were providedat the amount of 5×10⁵ cells/ml, and 24 hours after, when the number ofthe cells reaches 1×10⁶ cells/ml, the temporary expression wasperformed. A transfection was performed by liposomal reagent methodusing Freestyle™ MAX reagent (Invitrogen). In 15 ml tube, the DNAfragments encoding the heavy chain and the light chain were provided atthe ratio of 1:1 (heavy chain DNA: light chain DNA), and mixed with 2 mlof OptiPro™ SFM (Invtrogen) (mixture (A)). In another 15 ml tube, 100 mlof Freestyle™ MAX reagent and 2 ml of OptiPro™ SFM 2 ml were mixed(mixture (B)). Then mixtures (A) and (B) were mixed and incubated for 15minutes, and the obtained mixture was slowly added to and mixed with thecell provided one day before. After the transfection was completed, thecells were incubated for 4 days under the conditions of 37° C., 80%humidity, and 8% CO₂ in 130 rpm incubator.

The incubated cells were centrifuged and 100 ml of each supernatant wascollected and purified using AKTA Prime (GE Healthcare). AKTA Prime wasequipped with HiTrap MabSelect SuRe column (GE Healthcare, 11-0034-95).The cell culture solution was flowed at the flow rate of 5 ml/min, andthen, eluted with IgG elution buffer (Thermo Scientific, 21004), and theobtained eluate was exchanged with PBS buffer.

Finally, an antibody wherein the anti-Ang2 scFv is fused to theC-terminus of the anti-c-Met antibody L3-1Y-IgG2 was obtained and namedas an anti-c-Met/anti-Ang2 bispecific antibody (referred to in thefigures as “BS ab”).

Example 2 Binding Affinity of the anti-c-Met/anti-Ang2 BispecificAntibody to c-Met

The binding affinity of the anti-c-Met/anti-Ang2 bispecific antibodyprepared in Example 1 to c-Met was measured using Biacore T100 (GE). Forthis, a human Fab binder (GE Healthcare) was fixed on the surface of CMSchip (#BR-1005-30, GE) according to manufacturer's manual. About 90˜120RU of the anti-c-Met/anti-Ang2 bispecific antibody was captured andthen, c-Met-Fc (#358-MT/CF, R&D Systems) was added to the capturedantibody at various concentrations. Hereto, 10 mM Glycine-HCl (pH 1.5)solution was added, to regenerate the surface. To measure the affinity,the above obtained data were fitted using BIAevaluation software (GEHealthcare, Biacore T100 evaluation software).

The obtained results are illustrated in Table 20:

TABLE 20 R_(max) K_(D) k_(a) U- (RU) (nM) (l/Ms) k_(d) (l/s) Chi² ValueT (k_(a)) T (k_(d)) 65.40 0.09 8.5 × 10⁵ 8.2 × 10⁻⁵ 4.04 15 9.1 × 10² 54

As shown in Table 20, the bispecific antibody prepared in Example 1shows the high affinity (about 0.09 nM) to c-Met.

Example 3 Binding Affinity of the anti-c-Met/anti-Ang2 BispecificAntibody to Ang2

An ELISA for measuring the binding affinity of the anti-c-Met/anti-Ang2bispecific antibody prepared in Example 1 to Ang2 was performed. 96-wellMaxiSorp™ flat-bottom plate (Nunc) was coated with 1 μg/μl of human Ang2(R&D Systems) at the amount of 50 ul/well. Thereafter, the plate waswashed 5 times with 0.05% Tween-20-containing phosphate buffer saline(PBS) and blocked with 1% BSA-containing PBS for 2 hours at roomtemperature. The anti-c-Met/anti-Ang2 bispecific antibody prepared inExample 1 was added to the Ang2-coated well, and allowed to react for 1hour at room temperature. The anti-c-Met/anti-Ang2 bispecific antibodywas used by serially diluting from 10⁴ nM (see FIG. 3). Then, the wellwas washed 5 times with 0.05% Tween-20-containing PBS, and then, reactedwith HRP-conjugated anti-Human Fc antibody (Sigma) that is diluted with1% BSA-containing PBS to the ratio of 1:5,000, for 1 hour at roomtemperature, and then washed 5 times with 0.1% Tween-20-containing PBS.Finally, 100 μl of TMB substrate (SIGMA) was added to each well of theplate, to induce coloring response. Then, the response was stopped using50 μl of 5N H₂SO₄ solution. The value of OD450 was measured on a platereader (Molecular Devices). The binding affinity to Ang2 was obtained bydetermining the 50% binding concentrations to human Ang2 protein fromthe above results. The obtained results are illustrated in Table 21 andFIG. 3:

TABLE 21 Kd (nM) BS Ab 31 Anti-Ang2 Ab (4-H10) 0.035 (BS ab: theanti-c-Met/anti-Ang2 bispecific antibody of Example 1)

As shown in Table 21 and FIG. 3, the bispecific antibody prepared inExample 1 has binding affinity to Ang2 of about 31 nM.

Example 4 Growth Inhibition of Vascular Cells by theAnti-c-Met/anti-Ang2 Bispecific Antibody

To verify the effect of the anti-c-Met/anti-Ang2 bispecific antibodyprepared in Example 1 on vascular cell growth, Cell Counting Kit-8(CCK-8, supplied by Dojinho Molecular Technologies, Inc. Cat. #CK04-01)was used.

CCK-8 enables sensitive colorimetric assay, thereby achieving an exactmeasurement of cell viability in cell proliferation and cytotoxicityassay. A highly water-soluble tetrazolium salt, WST-8[2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium,monosodium salt; referring to the following chemical formula] is reducedby a dehydrogenase activity in a cell to be yellow formazan dye whichcan be dissolved in a tissue culturing medium.

The amount of formazan dye generated by dehydrogenase activity in a cellis direct proportional to the number of living cells, and thus, the cellviability can be determined by measuring the amount of formazan dye. Inthis example, it was tested how much the anti-c-Met/anti-Ang2 bispecificantibody or each inhibitor inhibits the cell growth which is increasedby treating Ang2 and c-Met ligand, HGF/SF.

In particular, the vascular cell growth inhibition effect of theanti-c-Met/anti-Ang2 bispecific antibody prepared in Example 1 wastested in human umbilical vein endothelial cells (HUVEC) and lymphaticendothelial cells (LEC). The HUVEC cells were obtained from ATCC and theLEC cells were obtained from Lonza.

The cells were cultured in EGM-2 medium (Lonza) under the conditions of5% CO₂ and 37° C. For a cell proliferation assay, the cells (5000cells/well, 0.5% FBS in EBM2) were subcultured in 96 well plate, whereinthe cells were treated with the anti-c-Met/anti-Ang2 bispecific antibodyprepared in Example 1 at the amount of 2μg/ml (40 nM) and cultured for72 hours. 100 μg/ml of HGF (#294-HG/CF, R&D SYSTEMS, Minneapolis, Minn.)was treated to the culture together. As a negative control, a mediumwhich is not treated with the antibody was used, and as positivecontrols, groups treated with 4-H10 anti-Ang2 antibody prepared inReference Example 2, treated with L3-1Y-IgG2 antibody prepared inReference Example 1, and co-treated with L3-1Y-IgG2 antibody and 4-H10antibody were used, wherein each antibody was used at the amount of 60nM (in the case of co-treatment, each antibody was treated the amount of60 nM).

After the culture, the degree of cell proliferation was analyzed usingCell Counting Kit-8 assay (Dojindo Molecular Technologies, Gaithersburg,Md.) according to the manufacturer's manual. In brief, after the 72 hourculture, CCK8 solution was added to each well at the amount of 10 μl,and then, the cells were further cultured for 2.5 hours. Then, theabsorbance at 450 nm was measured with microplate reader.

The obtained results were illustrated in FIG. 4 (for HUVEC) and FIG. 5(for LEC). As shown in FIGS. 4 and 5, the anti-c-Met/anti-Ang2bispecific antibody exhibits cell proliferation inhibitory effect whichis considerable increased compared with the anti-c-Met antibodyL3-1Y-IgG2 or the anti-Ang2 antibody 4-H10, and which is equal to orhigher than that of the co-treatment of the anti-c-Met antibodyL3-1Y-IgG2 or the anti-Ang2 antibody 4-H10.

Example 5 Inhibition of Cell Migration by the Anti-c-Met/anti-Ang2Bispecific Antibody

Since Ang2 and c-Met are both related to cancer metastasis as well ascancer cell proliferation, the effect of the anti-c-Met/anti-Ang2bispecific antibody prepared in Example 1 on cell motility was observed.The inhibition effect of the bispecific antibody on HUVEC migration byAng2+HGF was tested as follows:

The motility of endothelial cells was measured using xCelligence RTCA(Realtime cell analyzer; GE Healthcare). The RTCA is a non-invasive cellmonitoring system capable of measuring impedance in real-time therebydetermining changes of cells. For the cell migration assay, CIM-plate16(GE Healthcare) consisting of a lower chamber and a upper chamber wasused, wherein microelectrodes for measuring the impedance are arrangedon the upper chamber, whereby when the cells seeded on the chambermigrate through micropores, the migration degree of cells attached tothe microelectrodes can be confirmed. The migration degree was convertedas a migration index. HUVEC cells (HUVEC P3-6, ATCC) which were grown inEGM-2 medium (Lonza) were incubated in 1% FBS-containing EBM medium for6 hours.

To each well of the lower chamber of the CIM-plate16 including 2%FBS-containing EBM medium, HGF 100 ng/ml and Ang2 2 ug/ml were addedtogether with 4H10 antibody (Reference Example 2), L3-1Y-IgG1 antibody(Reference Example 1), a combination of 4H10 and L3-1Y-IgG1, or theanti-c-Met/anti-Ang2 bispecific antibody (Example 1), wherein eachantibody was used at the concentration of 100 nM (in the case ofcombination, each antibody was used at the concentration of 100 nM), andthen, assembled with the upper chamber coated with fibronectin (Sigma).Serum free EBM medium was added to the upper chamber at the amount of 30μl, and then, left in incubator for 1 hour for equilibration between theplate and the medium. The CIM-plate was equipped in a device station inthe incubator and then, the background value was measured.

The incubated HUVEC cells were re-suspended with EBM-2 Serum-free media(Lonza), seeded at the amount of 60,000 cells/well, left for 15 minutesto be settle down, and then equipped in the device, to measure the cellmigration degree in real-time. The cell migration degree was expressedas Slope (1/hr).

The obtained results are illustrated in FIG. 6. As shown in FIG. 6, theanti-c-Met/anti-Ang2 bispecific antibody exhibits an inhibitory effecton HUVEC migration by Ang2+HGF wherein the inhibitory effect isconsiderably increased compared with that of L3-1Y-IgG2 or 4-H10 alone,and equal to or higher than that of a combination of L3-1Y-IgG2 and4-H10.

While one or more embodiments of the present invention have beendescribed with reference to the figures, it will be understood by thoseof ordinary skill in the art that various changes in form and detailsmay be made therein without departing from the spirit and scope of thepresent invention as defined by the following claims.

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and “at least one” andsimilar referents in the context of describing the invention (especiallyin the context of the following claims) are to be construed to coverboth the singular and the plural, unless otherwise indicated herein orclearly contradicted by context. The use of the term “at least one”followed by a list of one or more items (for example, “at least one of Aand B”) is to be construed to mean one item selected from the listeditems (A or B) or any combination of two or more of the listed items (Aand B), unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

What is claimed is:
 1. An anti-c-Met/anti-Ang2 bispecific antibodycomprising: (a) an anti-c-Met antibody or an antigen-binding fragmentthereof and (b) an anti-Ang2 antibody or an antigen-binding fragmentthereof, wherein the anti-c-Met antibody or an antigen-binding fragmentthereof binds to an epitope comprising 5 to 19 contiguous amino acids ofSEQ ID NO: 71 comprising SEQ ID NO: 73; and the anti-Ang2 antibody or anantigen-binding fragment thereof is (i) an antibody or anantigen-binding fragment thereof that binds to human Ang2 (SEQ ID NO:172) in the region of the 417^(th) to 434^(th) amino acids of SEQ ID NO:172 (loop 1 of human Ang2), the 447^(th) to 454^(th) amino acids of SEQID NO: 172 (loop 2 of human Ang2), the 460^(th) to 468^(th) amino acidsof SEQ ID NO: 172 (loop 3 of human Ang2) or an amino acid sequence ofabout 2 to about 20 consecutive amino acids within SEQ ID NO: 172 thatcomprises at least one exposed amino acid residue of loop 1, loop 2, orloop 3; or (ii) an antibody or an antigen-binding fragment thereof thatcompetes with the antibody or an antigen-binding fragment of (i) forbinding to human Ang2.
 2. The anti-c-Met/anti-Ang2 bispecific antibodyof claim 1, wherein the anti-Ang2 antibody or an antigen-bindingfragment thereof binds to an epitope comprising at least one amino acidselected from the group consisting of I434, A449, P452, and N467 of SEQID NO: 172, or any combination thereof.
 3. The anti-c-Met/anti-Ang2bispecific antibody of claim 1, wherein the anti-Ang2 antibody or anantigen-binding fragment thereof comprises: at least one heavy chaincomplementarity determining region selected from the group consisting ofa polypeptide (CDR-H1) comprising SEQ ID NO: 151, a polypeptide (CDR-H2)comprising SEQ ID NO: 152, and a polypeptide (CDR-H3) comprising anamino acid sequence selected from the group consisting of SEQ ID NOs:122 to 127; at least one light chain complementarity determining regionselected from the group consisting of a polypeptide (CDR-L1) comprisingSEQ ID NO: 153, a polypeptide (CDR-L2) comprising SEQ ID NO: 154, and apolypeptide (CDR-L3) comprising SEQ ID NO: 155; or a combination of theat least one heavy chain complementarity determining region and the atleast one light chain complementarity determining region.
 4. Theanti-c-Met/anti-Ang2 bispecific antibody of claim 3, wherein theanti-Ang2 antibody or an antigen-binding fragment thereof comprises: atleast one heavy chain complementarity determining region selected fromthe group consisting of a polypeptide (CDR-H1) comprising an amino acidsequence selected from SEQ ID NOs: 109 to 112, a polypeptide (CDR-H2)comprising an amino acid sequence selected from SEQ ID NOs: 115 to 119,and a polypeptide (CDR-H3) comprising an amino acid sequence selectedfrom SEQ ID NOs: 122 to 127 at least one light chain complementaritydetermining region selected from the group consisting of a polypeptide(CDR-L1) comprising an amino acid sequence selected from SEQ ID NOs: 130to 135, a polypeptide (CDR-L2) comprising an amino acid sequenceselected from SEQ ID NOs: 138 to 142, and a polypeptide (CDR-L3)comprising an amino acid sequence selected from SEQ ID NOs: 145 to 148;or a combination of the at least one heavy chain complementaritydetermining region and the at least one light chain complementaritydetermining region.
 5. The anti-c-Met/anti-Ang2 bispecific antibody ofclaim 4, wherein the anti-Ang2 antibody or an antigen-binding fragmentthereof comprises: a heavy chain variable region comprising an aminoacid sequence selected from the group consisting of SEQ ID NOs: 156 to161; a light chain variable region comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 164 to 169; or acombination of the heavy chain variable region and the light chainvariable region.
 6. The anti-c-Met/anti-Ang2 bispecific antibody ofclaim 1, wherein the anti-c-Met antibody or an antigen-binding fragmentthereof comprises: (i) at least one heavy chain complementaritydetermining region (CDR) selected from the group consisting of (a) aCDR-H1 comprising SEQ ID NO: 4; (b) a CDR-H2 comprising SEQ ID NO: 5,SEQ ID NO: 2, or 8-19 contiguous amino acids within of SEQ ID NO: 2including the 3^(rd) to 10^(th) positions of SEQ ID NO: 2; and (c) aCDR-H3 comprising SEQ ID NO: 6, SEQ ID NO: 85, or 6-13 consecutive aminoacids within SEQ ID NO: 85 including the 1^(st) to 6^(th) positions ofSEQ ID NO: 85; (ii) at least one light chain complementarity determiningregion (CDR) selected from the group consisting of (a) a CDR-L1comprising SEQ ID NO: 7, (b) a CDR-L2 comprising SEQ ID NO: 8, and (c) aCDR-L3 comprising SEQ ID NO: 9, the amino acid sequence of SEQ ID NO:15, the amino acid sequence of SEQ ID NO: 86, or 9-17 consecutive aminoacids within SEQ ID NO: 89 including the 1^(st) to 9^(th) positions ofSEQ ID NO: 89; or (iii) a combination of the at least one heavy chaincomplementarity determining region and at least one light chaincomplementarity determining region.
 7. The anti-c-Met/anti-Ang2bispecific antibody of claim 6, wherein the CDR-H1 comprises SEQ ID NO:1, SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24, the CDR-H2 comprisesSEQ ID NO: 2, SEQ ID NO: 25, or SEQ ID NO: 26, the CDR-H3 comprises SEQID NO: 3, SEQ ID NO: 27, SEQ ID NO: 28, or SEQ ID NO: 85, the CDR-L1comprises SEQ ID NO: 10, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31,SEQ ID NO: 32, SEQ ID NO: 33, or SEQ ID NO: 106, the CDR-L2 comprisesSEQ ID NO: 11, SEQ ID NO: 34, SEQ ID NO: 35, or SEQ ID NO: 36, and theCDR-L3 comprises SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO:15, SEQ ID NO: 16, SEQ ID NO: 37, SEQ ID NO: 86, or SEQ ID NO:
 89. 8.The anti-c-Met/anti-Ang2 bispecific antibody of claim 6, wherein theanti-c-Met antibody or an antigen-binding fragment thereof comprises:(i) a heavy chain variable region comprising (a) a polypeptide (CDR-H1)comprising SEQ ID NO: 1, 22, 23, or 24, (b) a polypeptide (CDR-H2)comprising SEQ ID NO: 2, 25, or 26, and (c) a polypeptide (CDR-H3)comprising SEQ ID NO: 3, 27, 28, or 85; and (ii) a light chain variableregion comprising (a) a polypeptide (CDR-L1) comprising an SEQ ID NO:10, 29, 30, 31, 32, 33 or 106, (b) a polypeptide (CDR-L2) comprising SEQID NO: 11, 34, 35, or 36, and (c) a polypeptide (CDR-L3) comprising SEQID NO: 12, 13, 14, 15, 16, 37, 86, or
 89. 9. The anti-c-Met/anti-Ang2bispecific antibody of claim 1, comprising an anti-c-Met antibody thatcomprises two heavy chains and two light chains and an scFv of theanti-Ang2 antibody linked to the C-terminus of the anti-c-Met antibody.10. An anti-c-Met/anti-Ang2 bispecific antibody comprising an anti-c-Metantibody or an antigen-binding fragment thereof and an anti-Ang2antibody or an antigen-binding fragment thereof, wherein the anti-c-Metantibody or an antigen-binding fragment thereof binds to an epitopecomprising 5 to 19 contiguous amino acids of SEQ ID NO: 71 comprisingSEQ ID NO: 73, and the anti-Ang2 antibody or an antigen-binding fragmentthereof is (a) an antibody or an antigen-binding fragment thereofcomprising at least one heavy chain complementarity determining regionselected from the group consisting of a polypeptide (CDR-H1) comprisingSEQ ID NO: 113 or 114; a polypeptide (CDR-H2) comprising SEQ ID NO: 120or 121, and a polypeptide (CDR-H3) comprising SEQ ID NO: 128 or 129; atleast one light chain complementarity determining region selected fromthe group consisting of a polypeptide (CDR-L1) comprising SEQ ID NO: 136or 137, a polypeptide (CDR-L2) comprising SEQ ID NO: 143 or 144, and apolypeptide (CDR-L3) comprising SEQ ID NO: 149 or to 150; or acombination of the at least one heavy chain complementarity determiningregion and the at least one light chain complementarity determiningregion.
 11. The anti-c-Met/anti-Ang2 bispecific antibody of claim 10,wherein the anti-Ang2 antibody or an antigen-binding fragment thereofcomprises: a heavy chain variable region comprising SEQ ID NO: 162 or163; a light chain variable region comprising SEQ ID NO: 170 or 171; ora combination of the heavy chain variable region and the light chainvariable region.
 12. The anti-c-Met/anti-Ang2 bispecific antibody ofclaim 10, wherein the anti-c-Met antibody or an antigen-binding fragmentthereof comprises: (i) at least one heavy chain complementaritydetermining region (CDR) selected from the group consisting of (a) aCDR-H1 comprising SEQ ID NO: 4; (b) a CDR-H2 comprising SEQ ID NO: 5,the amino acid sequence of SEQ ID NO: 2, or 8-19 contiguous amino acidswithin SEQ ID NO: 2 including the 3^(rd) to 10^(th) positions of SEQ IDNO: 2; and (c) a CDR-H3 comprising SEQ ID NO: 6, SEQ ID NO: 85, or 6-13consecutive amino acids within SEQ ID NO: 85 including amino acidresidues from the 1^(st) to 6^(th) positions of SEQ ID NO: 85, (ii) atleast one light chain complementarity determining region (CDR) selectedfrom the group consisting of (a) a CDR-L1 comprising SEQ ID NO: 7, (b) aCDR-L2 comprising SEQ ID NO: 8, and (c) a CDR-L3 comprising SEQ ID NO:9, SEQ ID NO: 86, or 9-17 consecutive amino acids within SEQ ID NO: 89including the 1^(st) to 9^(th) positions of SEQ ID NO: 89; or (iii) acombination of the at least one heavy chain complementarity determiningregion and at least one light chain complementarity determining region.13. The anti-c-Met/anti-Ang2 bispecific antibody of claim 12, whereinthe CDR-H1 of the anti-c-Met antibody or an antigen-binding fragmentthereof comprises SEQ ID NO: 1, SEQ ID NO: 22, SEQ ID NO: 23, or SEQ IDNO: 24, the CDR-H2 of the anti-c-Met antibody or an antigen-bindingfragment thereof comprises SEQ ID NO: 2, SEQ ID NO: 25, or SEQ ID NO:26, the CDR-H3 of the anti-c-Met antibody or an antigen-binding fragmentthereof comprises SEQ ID NO: 3, SEQ ID NO: 27, SEQ ID NO: 28, or SEQ IDNO: 85, the CDR-L1 of the anti-c-Met antibody or an antigen-bindingfragment thereof comprises SEQ ID NO: 10, SEQ ID NO: 29, SEQ ID NO: 30,SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, or SEQ ID NO: 106, theCDR-L2 of the anti-c-Met antibody or an antigen-binding fragment thereofcomprises SEQ ID NO: 11, SEQ ID NO: 34, SEQ ID NO: 35, or SEQ ID NO: 36,and the CDR-L3 of the anti-c-Met antibody or an antigen-binding fragmentthereof comprises SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ IDNO: 15, SEQ ID NO: 16, SEQ ID NO: 37, SEQ ID NO: 86, or SEQ ID NO: 89.14. The anti-c-Met/anti-Ang2 bispecific antibody of claim 12, whereinthe anti-c-Met antibody or an antigen-binding fragment thereofcomprises: (i) a heavy chain variable region comprising (a) apolypeptide (CDR-H1) comprising SEQ ID NO: 1, 22, 23, or 24, (b) apolypeptide (CDR-H2) comprising SEQ ID NO: 2, 25, or 26, and (c) apolypeptide (CDR-H3) comprising SEQ ID NO: 3, 27, 28, or 85; and (ii) alight chain variable region comprising (a) a polypeptide (CDR-L1)comprising SEQ ID NO: 10, 29, 30, 31, 32, 33 or 106, (b) a polypeptide(CDR-L2) comprising SEQ ID NO: 11, 34, 35, or 36, and (c) a polypeptide(CDR-L3) comprising SEQ ID NO: 12, 13, 14, 15, 16, 37, 86, or
 89. 15.The anti-c-Met/anti-Ang2 bispecific antibody of claim 10, comprising ananti-c-Met antibody that comprises two heavy chains and two light chainsand an scFv of the anti-Ang2 antibody linked to the C-terminus of theanti-c-Met antibody.
 16. A pharmaceutical composition comprising theanti-c-Met/anti-Ang2 bispecific antibody of claim 1 and apharmaceutically acceptable carrier.
 17. A pharmaceutical compositioncomprising the anti-c-Met/anti-Ang2 bispecific antibody of claim 10 anda pharmaceutically acceptable carrier.
 18. A method of preventing ortreating a disease associated with c-Met or Ang2 in a subject, themethod comprising administering the anti-c-Met/anti-Ang2 bispecificantibody of claim 1 to the subject, wherein the a disease associatedwith c-Met or Ang2 is cancer, cancer metastasis, an eye disease, aninflammatory disease, an infection, a cardiovascular disease, a renaldisease, sepsis, asthma, edema, or hereditary hemorrhagictelangiectasia.
 19. A method of preventing or treating a diseaseassociated with c-Met or Ang2 in a subject, the method comprisingadministering the anti-c-Met/anti-Ang2 bispecific antibody of claim 10to the subject, wherein the a disease associated with c-Met or Ang2 isselected from a cancer, cancer metastasis, an eye disease, aninflammatory disease, infection, a cardiovascular disease, a renaldisease, sepsis, asthma, edema, and hereditary hemorrhagictelangiectasia.